Compositions and methods for the depletion of cd137+ cells

ABSTRACT

The invention provides methods of preventing and treating graft-versus-host-disease and autoimmune diseases, such as those arising from transplant therapy, by selective depletion of hematopoietic cells through the use of antibody-drug conjugates and ligand-drug conjugates that specifically bind CD137. The compositions and methods described herein can be used to treat a variety of pathologies, including stem cell disorders and other blood conditions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/875,793, filed on Jan. 19, 2018, which in turn claims priority toU.S. Provisional Application No. 62/448,741, filed on Jan. 20, 2017, andU.S. Provisional Application No. 62/595,977, filed on Dec. 7, 2017. Thecontents of the aforementioned applications are incorporated byreference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 21, 2018, isnamed M103034_1030US_C1_SL.txt and is 20,472 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of transplant therapy andprovides methods for the treatment of autoimmune diseases andgraft-versus-host disease (GVHD) by administration of antibodies,antibody-drug conjugates, and ligand-drug conjugates capable of bindingan antigen expressed by hematopoietic cells.

BACKGROUND OF THE INVENTION

While hematopoietic stem cells have significant therapeutic potential, alimitation that has hindered their use in the clinic has been thedevelopment of graft-versus-host disease (GVHD) some days or weeks afterthe cell transplant. While significant advances have been made withregard to the treatment of GVHD following transplantation, there isstill a need in the art for improved methods, particularly with respectto reducing mortality rates from GVHD. Conventional treatment of GVHDrequires systemic immunosuppressive therapy with potent drugs such ascorticosteroids and cyclosporine. Agents such as mycophenolate mofetil,rapamycin (sirolimus), imatinib, and rituximab are used in patients withsteroid-refractory GVHD. However, these treatments have limited efficacyand often cause severe adverse effects. Only 50% of patients with GVHDare able to discontinue immunosuppressive treatment within 5 years afterdiagnosis, and 10% require continued treatment beyond 5 years. Theremaining 40% die or develop recurrent malignancy before GVHD resolves.Five year survival rates of patients with high risk GVHD (plateletcounts <100,000/microliter or progressive onset from GVHD) is only40-50%. Thus, the development of innovative strategies to prevent andtreat GVHD represents an important unmet clinical need.

Like, GVHD, autoimmune diseases such as multiple sclerosis, rheumatoidarthritis, intestinal bowel disease, psoriasis, lupus, and Type 1diabetes are characterized by an abnormal immune response directedagainst normal self tissues. Autoimmune diseases are characterized byproduction of autoreactive T cells and antibodies reactive with hosttissues (autoantibodies). Traditional therapies for autoimmune diseaseinclude immunosuppressive agents that globally dampen immune responses.The benefits of such agents are often tempered by susceptibility toopportunistic infections, long-term risk of malignancy, toxicity andother unfavorable side effects. Thus, there is a need to develop astrategy to more specifically target the cellular mediators of both GVHDand autoimmune diseases.

SUMMARY OF THE INVENTION

The present invention provides methods for preventing and treating acuteand chronic forms of graft-versus-host disease (GVHD) and autoimmunediseases in a patient such as a human patient, receiving hematopoieticstem cell transplant therapy so as to reduce the morbidity and mortalityassociated with GVHD and autoimmune diseases. The invention additionallyfeatures methods of treating a variety of hematopoietic conditions, suchas sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrichsyndrome, adenosine deaminase deficiency-severe combinedimmunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemiaand Schwachman-Diamond syndrome, human immunodeficiency virus infection,and acquired immune deficiency syndrome, among others. The inventionfeatures methods of treating a patient with antibodies, antibody-drugconjugates, ligands, and ligand-drug conjugates capable of bindingproteins expressed by hematopoietic cells, such as CD137, so as todeplete a population of hematopoietic cells, such as T cells, within thepatient. This selective depletion of T cells in turn improves overalland relapse-free patient survival while significantly decreasing GVHDand autoimmune diseases.

In a first aspect, the invention features a method of treating GVHD in ahuman patient in need thereof by administering to the patient aneffective amount of an antibody, or antigen-binding fragment thereof, oran antibody drug conjugate (ADC), capable of binding CD137, wherein theantibody or antigen-binding fragment thereof is conjugated to acytotoxin via a linker.

In a second aspect, the invention provides a method of depleting apopulation of CD137+ cells in a human patient suffering from or at riskfor GVHD by administering to the patient an effective amount of anantibody, or antigen-binding fragment thereof, or an antibody drugconjugate (ADC), capable of binding CD137, wherein the antibody orantigen-binding fragment thereof is conjugated to a cytotoxin via alinker.

In a third aspect, the invention features a method of treating anautoimmune disease in a human patient in need thereof by administeringto the patient an effective amount of an antibody, or antigen-bindingfragment thereof, or antibody drug conjugate (ADC), capable of bindingCD137, wherein the antibody or antigen-binding fragment thereof isconjugated to a cytotoxin via a linker.

In a fourth aspect, the invention provides a method of depleting apopulation of CD137+ cells in a human patient suffering from or at riskfor an autoimmune disease by administering to the patient an effectiveamount of an antibody, or antigen-binding fragment thereof, or anantibody drug conjugate (ADC), capable of binding CD137, wherein theantibody or antigen-binding fragment thereof is conjugated to acytotoxin via a linker.

In some embodiments, the antibody, or antigen-binding fragment thereofis selected from the group consisting of a monoclonal antibody orantigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, anantibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem di-scFv.

In some embodiments, the antibody, or antigen-binding fragment thereofbinds human CD137 extracellular domain at an epitope located withinamino acid residues 115-156 of SEQ ID NO: 20. SEQ ID NO: 20 correspondsto the extracellular domain of CD137 and has the following amino acidsequence:

(SEQ ID NO: 20) MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL

In some embodiments, the antibody has an isotype selected from the groupconsisting of IgG, IgA, IgM, IgD, and IgE.

In some embodiments, the Fc domain is a human IgG1 isotype Fc domain. Insome embodiments, the Fc domain is a human IgG2 isotype Fc domain. Insome embodiments, the Fc domain is a human IgG3 isotype Fc domain. Insome embodiments, the Fc domain is a human IgG4 isotype Fc domain.

In another aspect, the invention features a method of treating GVHD in ahuman patient in need thereof, the method comprising administering tothe patient an effective amount of a soluble CD137 ligand.

In another aspect, the invention features a method of depleting apopulation of CD137 positive cells in a human patient suffering from orat risk for GVHD, the method comprising administering to the patient aneffective amount of a soluble CD137 ligand.

In another aspect, the invention features a method of treating anautoimmune disease in a human patient in need thereof, by administeringto the patient an effective amount of a soluble CD137 ligand.

In another aspect, the invention features a method of depleting apopulation of CD137 positive cells in a human patient suffering from orat risk for an autoimmune disease, by administering to the patient aneffective amount of a soluble CD137 ligand.

In some embodiments, the antibody or antigen-binding fragment thereof isconjugated to a cytotoxin, such as a microtubule-binding agent. In someembodiments, the antibody or antigen-binding fragment thereof, orsoluble CD137 ligand is conjugated to a microtubule-binding agent by wayof a linker. In yet other embodiments, the antibody or antigen-bindingfragment thereof is conjugated to a cytotoxin, wherein the cytotoxin isa microtubule-binding agent.

In some embodiments, the microtubule-binding agent is a maytansine.

In some embodiments, the microtubule-binding agent is a maytansinoid.

In some embodiments, the microtubule-binding agent is a maytansine or amaytansinoid.

In some embodiments, the maytansinoid is selected from the groupconsisting of DM1, DM3, and DM4, and maytansinol.

In some embodiments, the maytansinoid is a maytansinol analog.

In some embodiments, the antibody, antigen-binding fragment thereof,ADC, or soluble CD137 ligand is delivered into the patient prior to thepatient receiving a transplant comprising hematopoietic stem cells.

In some embodiments, the antibody, antigen-binding fragment thereof,ADC, or soluble CD137 ligand conjugated to a cytotoxin, such as amicrotubule binding agent, is delivered into the patient about 3 days(for example, from 1 hour to 7 days (e.g., 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days,4 days, 5 days, 6 days, or 7 days) prior to administration of thehematopoietic stem cells into the patient.

In some embodiments, the antibody, antigen-binding fragment thereof,ADC, or soluble CD137 ligand is delivered into the patient concomitantwith the patient receiving a transplant that includes hematopoietic stemcells.

In some embodiments, the antibody, antigen-binding fragment thereof,ADC, or soluble CD137 ligand is delivered into the patient after thepatient receives a transplant comprising hematopoietic stem cells.

In some embodiments, the antigen-binding fragment thereof, ADC, orsoluble CD137 ligand (e.g., conjugated to a cytotoxin, such as amicrotubule binding agent) is delivered into the patient, for example,about 1 hour to 10 days (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, or 10 days) or more after theadministration of the exogenous hematopoietic stem cell transplant. Forexample, the antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate may be administered about 3 to 4days after the transplant.

In some embodiments, the transplant is allogeneic. In some embodiments,the transplant is autologous.

In some embodiments, the transplant is a bone marrow transplant, aperipheral blood transplant, or a cord blood transplant.

In some embodiments, the transplant includes hematopoietic cells (e.g.,hematopoietic stem cells).

In some embodiments, the hematopoietic stem cells or progeny thereofmaintain hematopoietic stem cell functional potential after two or moredays following transplantation of hematopoietic stem cells into thepatient.

In some embodiments, the hematopoietic stem cells or progeny thereofmaintain hematopoietic stem cell functional potential after two or moredays (for example, from about 2 to about 5 days, from about 2 to about 7days, from about 2 to about 20 days, from about 2 to about 30 days, suchas 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26days, 27 days, 28 days, 29 days, 30 days, or more) followingtransplantation of the hematopoietic stem cells into the patient.

In some embodiments, the hematopoietic stem cells or progeny thereof arecapable of localizing to hematopoietic tissue, such as the bone marrow,and/or reestablishing hematopoiesis following transplantation of thehematopoietic stem cells into the patient.

In some embodiments, upon transplantation into the patient, thehematopoietic stem cells give rise to recovery of a population of cellsselected from the group consisting of megakaryocytes, thrombocytes,platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils,eosinophils, microglia, granulocytes, monocytes, osteoclasts,antigen-presenting cells, macrophages, dendritic cells, natural killercells, T cells and B cells.

In some embodiments, the transplant comprises leukocytes.

In some embodiments, upon transplantation into the patient, thehematopoietic cells are selected from the group consisting of T cells, Bcells, dendritic cells, natural killer (NK) cells, macrophages, cancercells, neutrophils, basophils, and eosinophils.

In some embodiments, the invention provides a method of depleting apopulation of CD137+ cells in a human patient suffering from or at riskfor GVHD by administering to the patient an effective amount of anantibody, or antigen-binding fragment, ADC, or soluble CD137 ligandcapable of binding CD137 and is conjugated to a cytotoxin such as amicrotubule-binding agent, wherein the hematopoietic cells comprisingCD137+ cells are selected from the group consisting of T cells, B cells,dendritic cells, natural killer (NK) cells, macrophages, cancer cells,neutrophils, basophils, and eosinophils.

In some embodiments, CD137+ cells selected from the group consisting ofT cells, B cells, dendritic cells, natural killer (NK) cells,macrophages, cancer cells, neutrophils, basophils, and eosinophilsdemonstrate reactivity against an antigen of the patient.

In some embodiments, the antibody, antigen-binding fragment thereof,ADC, or soluble CD137 ligand is internalized by a CD137+ cell followingadministration to the patient. For instance, the antibody,antigen-binding fragment thereof, ADC, or soluble CD137 ligand may beinternalized by a CD137+ T cell by receptor mediated endocytosis (e.g.,upon binding to cell-surface CD137). In some embodiments, a cytotoxincovalently bound to the antibody, antigen-binding fragment thereof, orADC, may be released intracellularly by chemical cleavage (for instance,by enzymatic or non-specific cleavage of a linker described herein). Thecytotoxin may then access its intracellular target (such as the mitoticspindle apparatus, nuclear DNA, ribosomal RNA, or topoisomerases, amongothers) so as to promote the death of a CD137+ T cell.

In some embodiments, the antibody, antigen-binding fragment thereof,ADC, or soluble CD137 ligand is capable of promoting mitotic arrest andsuppressing proliferation (for instance, by suppressing microtubuledynamic instability) of the CD137+ T cell.

In some embodiments, the antibody, antigen-binding fragment thereof,ADC, or soluble CD137 ligand may promote the death of a cell byrecruiting one or more complement proteins, natural killer (NK) cells,macrophages, neutrophils, and/or eosinophils upon administration to thepatient.

In some embodiments, the antibody, antigen-binding fragment thereof,ADC, or soluble CD137 ligand may promote the death of a CD137+ T cell byrecruiting one or more complement proteins, natural killer (NK) cells,macrophages, neutrophils, and/or eosinophils upon administration to thepatient.

In some embodiments, the antibody or antigen-binding fragment thereof orsoluble CD137 ligand is used to treat a T- or B cell-driven autoimmunedisease.

In some embodiments, the autoimmune disease is multiple sclerosis,rheumatoid arthritis, intestinal bowel disease, psoriasis, lupus, orType 1 diabetes.

In some embodiments, the method is used to treat one or more disordersor cancers in a patient, such as a patient that has received atransplant comprising hematopoietic stem cells. For instance, thepatient may be one that is suffering from a stem cell disorder. In someembodiments, the patient is suffering from a hemoglobinopathy disorder,such as sickle cell anemia, thalassemia, Fanconi anemia, andWiskott-Aldrich syndrome. The patient may be suffering from animmunodeficiency disorder, such as a congenital immunodeficiencydisorder or an acquired immunodeficiency disorder (e.g., humanimmunodeficiency virus or acquired immune deficiency syndrome). In someembodiments, the patient is suffering from a metabolic disorder, such asglycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease,Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy. Insome embodiments, the patient is suffering from cancer, such asleukemia, lymphoma, multiple myeloma and myelodysplastic syndrome, andneuroblastoma. In some embodiments, the patient is suffering from adisorder selected from the group consisting of adenosine deaminasedeficiency and severe combined immunodeficiency, hyper immunoglobulin Msyndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis,osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemiamajor, systemic sclerosis, systemic lupus erythematosus, multiplesclerosis, and juvenile rheumatoid arthritis.

In a further aspect, the invention features a method of treating graftversus host disease (GVHD) in a human patient in need thereof, byadministering an anti-CD137 antibody drug conjugate (ADC) to the humanpatient such that GVHD is treated, wherein the ADC comprises ananti-CD137 antibody linked to a cytotoxin which is a microtubule-bindingagent or an RNA polymerase inhibitor. In one embodiment, the methodcomprises administering the ADC to the patient prior to the patientreceiving a transplant comprising hematopoietic stem cells. In anotherembodiment, the method comprising administering the ADC to the patientabout three days prior to the patient receiving a transplant comprisinghematopoietic stem cells. In another embodiment, the method comprisesadministering the ADC to the patient concomitant with the patientreceiving a transplant comprising hematopoietic stem cells. In a furtherembodiment, the method comprises administering the ADC to the patientafter the patient receives a transplant comprising hematopoietic stemcells. In yet another embodiment, the method comprises administering theADC to the patient about 1 hour to 10 days after the patient receives atransplant comprising hematopoietic stem cells. In a further embodiment,the method comprises administering the ADC to the patient about 3 to 4days after the patient receives a transplant comprising hematopoieticstem cells. In other embodiments, the transplant is allogeneic.

In yet another aspect, the invention features a method of depleting apopulation of CD137 positive cells in a human subject having GVHD or atrisk of developing GVHD, by administering an anti-CD137 ADC to the humanpatient such that GVHD the population of CD137 cells is depleted,wherein the ADC comprises an anti-CD137 antibody linked to a cytotoxinwhich is a microtubule-binding agent or an RNA polymerase inhibitor. Inone embodiment, the method comprises administering the ADC to thepatient prior to the patient receiving a transplant comprisinghematopoietic stem cells. In another embodiment, the method comprisesadministering the ADC to the patient about three days prior to thepatient receiving a transplant comprising hematopoietic stem cells. Inanother embodiment, the method comprises administering the ADC to thepatient concomitant with the patient receiving a transplant comprisinghematopoietic stem cells. In a further embodiment, the method comprisesadministering the ADC to the patient after the patient receives atransplant comprising hematopoietic stem cells. In yet anotherembodiment, the method comprises administering the ADC to the patientabout 1 hour to 10 days after the patient receives a transplantcomprising hematopoietic stem cells. In a further embodiment, the methodcomprises administering the ADC to the patient about 3 to 4 days afterthe patient receives a transplant comprising hematopoietic stem cells.In other embodiments, the transplant is allogeneic. In otherembodiments, the microtubule-binding agent is a maytansinoid. In otherembodiments, the RNA polymerase inhibitor is an amatoxin. In certainembodiments, the amatoxin is represented by formula (IA)

-   -   wherein R₁ is H, OH, OR_(A), or OR_(C);    -   R₂ is H, OH, OR_(B), or OR_(C);    -   R_(A) and R_(B), together with the oxygen atoms to which they        are bound, combine to form an optionally substituted 5-membered        heterocyclolalkyl group;    -   R₃ is H, R_(C), or R_(D);    -   R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D),        R_(C), or R_(D);    -   R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);    -   R₉ is H, OH, OR_(C), or OR_(D);    -   X is —S—, —S(O)—, or —SO₂—;    -   R_(C) is -L-Z;    -   R_(D) is optionally substituted C₁-C₆ alkyl, optionally        substituted C₁-C₆ heteroalkyl, optionally substituted C₂-C₆        alkenyl, optionally substituted C₂-C₆ heteroalkenyl, optionally        substituted C₂-C₆ alkynyl, optionally substituted C₂-C₆        heteroalkynyl, optionally substituted cycloalkyl, optionally        substituted heterocycloalkyl, optionally substituted aryl, or        optionally substituted heteroaryl;    -   L is optionally substituted C₁-C₆ alkylene, optionally        substituted C₁-C₆ heteroalkylene, optionally substituted C₂-C₆        alkenylene, optionally substituted C₂-C₆ heteroalkenylene,        optionally substituted C₂-C₆ alkynylene, optionally substituted        C₂-C₆ heteroalkynylene, optionally substituted cycloalkylene,        optionally substituted heterocycloalkylene, optionally        substituted arylene, or optionally substituted heteroarylene;        and    -   Z is a chemical moiety formed from a coupling reaction between a        reactive substituent present on L and a reactive substituent        present within the antibody or antigen-binding fragment thereof,

wherein Am comprises exactly one R_(C) substituent. In yet otherembodiments, the amatoxin is represented by formula (IB)

-   -   wherein R₁ is H, OH, OR_(A), or OR_(C);    -   R₂ is H, OH, OR_(B), or OR_(C);    -   R_(A) and R_(B), together with the oxygen atoms to which they        are bound, combine to form an optionally substituted 5-membered        heterocyclolalkyl group;    -   R₃ is H, R_(C), or R_(D);    -   R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D),        R_(C), or R_(D);    -   R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);    -   R₉ is H, OH, OR_(C), or OR_(D);    -   X is —S—, —S(O)—, or —SO₂—;    -   R_(C) is -L-Z;    -   R_(D) is optionally substituted C₁-C₆ alkyl, optionally        substituted C₁-C₆ heteroalkyl, optionally substituted C₂-C₆        alkenyl, optionally substituted C₂-C₆ heteroalkenyl, optionally        substituted C₂-C₆ alkynyl, optionally substituted C₂-C₆        heteroalkynyl, optionally substituted cycloalkyl, optionally        substituted heterocycloalkyl, optionally substituted aryl, or        optionally substituted heteroaryl;    -   L is optionally substituted C₁-C₆ alkylene, optionally        substituted C₁-C₆ heteroalkylene, optionally substituted C₂-C₆        alkenylene, optionally substituted C₂-C₆ heteroalkenylene,        optionally substituted C₂-C₆ alkynylene, optionally substituted        C₂-C₆ heteroalkynylene, optionally substituted cycloalkylene,        optionally substituted heterocycloalkylene, optionally        substituted arylene, or optionally substituted heteroarylene;        and    -   Z is a chemical moiety formed from a coupling reaction between a        reactive substituent present on L and a reactive substituent        present within the antibody or antigen-binding fragment thereof,    -   wherein Am comprises exactly one R_(C) substituent.

In other embodiments, the ADC comprises an anti-CD137 antibodyconjugated to a cytotoxin which is an RNA polymerase inhibitor, e.g., anamanitin. In one embodiment, the amanitin is α-amanitin, β-amanitin,γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanullinicacid, or proamanullin.

In another aspect, the invention features a method of depletingallo-reactive T cells in a human patient who received an allogenictransplant, by administering an anti-CD137 ADC to the human patient suchallo-reactive T cells are depleted, wherein the ADC comprises ananti-CD137 antibody linked to a cytotoxin. In certain embodiments, thetransplant is a bone marrow transplant. In other embodiments, thetransplant is a peripheral blood transplant. In yet other embodiments,the transplant is a cord blood transplant. In other embodiments, thetransplant comprises hematopoietic cells. In certain embodiments, thehematopoietic stem cells or progeny thereof maintain hematopoietic stemcell functional potential after two or more days followingtransplantation of the hematopoietic stem cells into the patient. Inother embodiments, the cytotoxin is an RNA polymerase inhibitor. Inanother embodiment, the RNA polymerase inhibitor is an amatoxin. Inother embodiments, the anti-CD137 antibody comprises the CDRs ofantibody BBK2 as defined by Kabat numbering. In another embodiment, theanti-CD137 antibody comprises the heavy and light chain variable regionsof antibody BBK2 (as described in Lee, et al. Eur J Immunogenet. 2002October; 29(5):449-52, incorporated herein by reference in itsentirety). In other embodiments, the anti-CD137 antibody is chimericBBK2. In other embodiments, the anti-CD137 antibody comprises a heavychain sequence comprising SEQ ID NO: 23 and a light chain sequencecomprising SEQ ID NO: 24. In yet other embodiments, the anti-CD137antibody is an antagonist antibody.

In other embodiments, the anti-CD137 antibody is an IgG1 or an IgG4. Incertain embodiments, the anti-CD137 antibody is an intact antibody.

In one embodiment, the microtubule-binding agent used in the methods andcompositions described herein is a maytansinoid.

In other embodiments, the RNA polymerase inhibitor used in the methodsand compositions described herein is an amatoxin. In certainembodiments, the amatoxin is represented by formula (IA)

-   -   wherein R₁ is H, OH, OR_(A), or OR_(C);    -   R₂ is H, OH, OR_(B), or OR_(C);    -   R_(A) and R_(B), together with the oxygen atoms to which they        are bound, combine to form an optionally substituted 5-membered        heterocyclolalkyl group;    -   R₃ is H, R_(C), or R_(D);    -   R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D),        R_(C), or R_(D);    -   R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);    -   R₉ is H, OH, OR_(C), or OR_(D);    -   X is —S—, —S(O)—, or —SO₂—;    -   R_(C) is -L-Z;    -   R_(D) is optionally substituted C₁-C₆ alkyl, optionally        substituted C₁-C₆ heteroalkyl, optionally substituted C₂-C₆        alkenyl, optionally substituted C₂-C₆ heteroalkenyl, optionally        substituted C₂-C₆ alkynyl, optionally substituted C₂-C₆        heteroalkynyl, optionally substituted cycloalkyl, optionally        substituted heterocycloalkyl, optionally substituted aryl, or        optionally substituted heteroaryl;    -   L is optionally substituted C₁-C₆ alkylene, optionally        substituted C₁-C₆ heteroalkylene, optionally substituted C₂-C₆        alkenylene, optionally substituted C₂-C₆ heteroalkenylene,        optionally substituted C₂-C₆ alkynylene, optionally substituted        C₂-C₆ heteroalkynylene, optionally substituted cycloalkylene,        optionally substituted heterocycloalkylene, optionally        substituted arylene, or optionally substituted heteroarylene;        and    -   Z is a chemical moiety formed from a coupling reaction between a        reactive substituent present on L and a reactive substituent        present within the antibody or antigen-binding fragment thereof,

wherein Am comprises exactly one R_(C) substituent. In yet otherembodiments, the amatoxin is represented by formula (IB)

-   -   wherein R₁ is H, OH, OR_(A), or OR_(C);    -   R₂ is H, OH, OR_(B), or OR_(C);    -   R_(A) and R_(B), together with the oxygen atoms to which they        are bound, combine to form an optionally substituted 5-membered        heterocyclolalkyl group;    -   R₃ is H, R_(C), or R_(D);    -   R₄, R₅, R₆, and R₇ are each independently H, OH, OR_(C), OR_(D),        R_(C), or R_(D);    -   R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);    -   R₉ is H, OH, OR_(C), or OR_(D);    -   X is —S—, —S(O)—, or —SO₂—;    -   R_(C) is -L-Z;    -   R_(D) is optionally substituted C₁-C₆ alkyl, optionally        substituted C₁-C₆ heteroalkyl, optionally substituted C₂-C₆        alkenyl, optionally substituted C₂-C₆ heteroalkenyl, optionally        substituted C₂-C₆ alkynyl, optionally substituted C₂-C₆        heteroalkynyl, optionally substituted cycloalkyl, optionally        substituted heterocycloalkyl, optionally substituted aryl, or        optionally substituted heteroaryl;    -   L is optionally substituted C₁-C₆ alkylene, optionally        substituted C₁-C₆ heteroalkylene, optionally substituted C₂-C₆        alkenylene, optionally substituted C₂-C₆ heteroalkenylene,        optionally substituted C₂-C₆ alkynylene, optionally substituted        C₂-C₆ heteroalkynylene, optionally substituted cycloalkylene,        optionally substituted heterocycloalkylene, optionally        substituted arylene, or optionally substituted heteroarylene;        and    -   Z is a chemical moiety formed from a coupling reaction between a        reactive substituent present on L and a reactive substituent        present within the antibody or antigen-binding fragment thereof,    -   wherein Am comprises exactly one R_(C) substituent. In other        embodiments, the RNA polymerase inhibitor is an amanitin. In yet        other embodiments, the amanitin is α-amanitin, β-amanitin,        γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin,        amanullinic acid, or proamanullin.

In one embodiment, featured is an antibody drug conjugate (ADC)comprising an anti-CD137 antibody conjugated to a cytotoxin via alinker. The cytotoxin can be, for example, an amanitin, e.g.,α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide,amanullin, amanullinic acid, and proamanullin. In one embodiment, thecytotoxin is α-amanitin. In one embodiment, the cytotoxin is β-amanitin.In one embodiment, the cytotoxin is γ-amanitin. In one embodiment, thecytotoxin is ε-amanitin. In one embodiment, the cytotoxin is amanin. Inone embodiment, the cytotoxin is amaninamide. In one embodiment, thecytotoxin is amanullin. In one embodiment, the cytotoxin is amanullinicacid. In one embodiment, the cytotoxin is proamanullin. In oneembodiment, the anti-CD137 antibody binds to the ectodomain of humanCD137. In one embodiment, the anti-CD137 antibody competes with antibodyBBK2.

In some embodiments, the anti-CD137 antibody comprises the CDRs ofantibody BBK2. In other embodiments, the anti-CD137 antibody comprisesthe heavy and light chain variable regions of antibody BBK2. In yetother embodiments, the anti-CD137 antibody is chimeric BBK2. In otherembodiments, the anti-CD137 antibody comprises a heavy chain sequencecomprising SEQ ID NO: 23 and a light chain sequence comprising SEQ IDNO: 24. In certain embodiments, the anti-CD137 antibody is an IgG1 or anIgG4.

In another aspect, the invention features an antibody, or an antigenbinding portion thereof, that specifically binds human CD137, saidantibody comprising a heavy chain comprising an amino acid sequence asset forth in SEQ ID NO: 23, and comprising a light chain comprising anamino acid sequence as set forth in SEQ ID NO: 24. In certainembodiments, the antibody, or an antigen binding portion thereof is anintact antibody.

In another aspect, the invention features antibody drug conjugate (ADC)comprising the anti-CD137 antibody, or antigen binding portion thereof,of the invention, wherein the antibody antigen binding portion thereof,is conjugated to a cytotoxin via a linker. In certain embodiments, thecytotoxin is a microtubule-binding agent or an RNA polymerase inhibitor.In other embodiments, the RNA polymerase inhibitor is an amatoxin. Inyet other embodiments, the amatoxin is an amanitin. In otherembodiments, the amanitin is α-amanitin, β-amanitin, γ-amanitin,ε-amanitin, amanin, amaninamide, amanullin, amanullinic acid, orproamanullin. The linker can be, for example, a dipeptide which isVal-Ala or Val-Cit. In some embodiments, the linker includes apara-aminobenzyl group (PAB). In some embodiments, the linker includesthe moiety PAB-Cit-Val. In some embodiments, the linker includes themoiety PAB-Ala-Val. In some embodiments, the linker includes a—((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6. In someembodiments, the linker is -PAB-Cit-Val-((C═O)(CH₂)_(n)—. In someembodiments, the linker is -PAB-Ala-Val-((C═O)(CH₂)_(n)—.

In another aspect, the invention features a pharmaceutical compositioncomprising an ADC of the invention, and a pharmaceutically activecarrier.

In another aspect, the invention features a method of treating graftfailure or GVHD in a human patient in need thereof, said methodcomprising administering an effective amount of an ADC of the inventionto the human patient, wherein the human patient previously received atransplant. In certain embodiments, the human patient received thetransplant no more than 4 days prior to the administration of the ADC.

In another aspect, the invention features a method of treating a humanpatient at risk for graft failure or GVHD, whereby an effective amountof an ADC described herein is administered to the human patient at riskof having graft failure or GVHD, followed by administration of atransplant to the human patient. In some embodiments, the ADC isadministered to the human patient as a single dose.

In another aspect, the invention features an ADC comprising ananti-CD137 antibody conjugated to a cytotoxin via a linker, where theantibody comprises a heavy chain variable region comprising a CDR1, aCDR2, and a CDR3 having an amino acid sequence as set forth in SEQ IDNOs: 25, 26, and 27, respectively, and comprising a light chain variableregion comprising a CDR1, a CDR2, and a CDR3 having an amino acidsequence as set forth in SEQ ID NOs: 29, 30, and 31, respectively. Insome embodiments, the antibody is a chimeric or a humanized antibody. Incertain embodiments, the heavy chain variable region comprises an aminoacid sequence as set forth in SEQ ID NO: 28 and the light chain variableregion comprises an amino acid sequence as set forth in SEQ ID NO: 32.In other embodiments, the antibody is an IgG1 or an IgG4 isotype. Inother embodiments, the cytotoxin is a microtubule-binding agent or anRNA polymerase inhibitor. In yet other embodiments, the RNA polymeraseinhibitor is an amatoxin. In yet other embodiments, the amatoxin is anamanitin. In other embodiments, the amanitin is selected from the groupconsisting of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin,amaninamide, amanullin, amanullinic acid, and proamanullin.

In another aspect, the invention features an antibody drug conjugate(ADC) comprising an anti-CD137 antibody conjugated to a cytotoxin via alinker, wherein the antibody comprises a heavy chain variable regioncomprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence asset forth in SEQ ID NOs: 33, 34, and 35, respectively, and comprising alight chain variable region comprising a CDR1, a CDR2, and a CDR3 havingan amino acid sequence as set forth in SEQ ID NOs: 36, 37, 28,respectively. In some embodiments, the antibody is a chimeric or ahumanized antibody. In other embodiments, the antibody is an IgG1 or anIgG4 isotype. In other embodiments, the cytotoxin is amicrotubule-binding agent or an RNA polymerase inhibitor. In yet otherembodiments, the RNA polymerase inhibitor is an amatoxin. In yet otherembodiments, the amatoxin is an amanitin. In other embodiments, theamanitin is α-amanitin, (3-amanitin, γ-amanitin, ε-amanitin, amanin,amaninamide, amanullin, amanullinic acid, or proamanullin.

In another aspect, the invention features a composition comprising anyof the ADCs described herein, and a pharmaceutically acceptable carrier.

In another aspect, the invention features a method of treating graftfailure or GVHD in a human patient in need thereof, by administering aneffective amount of an ADC as described herein to the human patient,wherein the human patient previously received a transplant. In someembodiments, the human patient received the transplant no more than 4days prior to the administration of the ADC. In some embodiments, theADC is administered to the human patient as a single dose.

In another aspect, the invention features a method of treating humanpatient at risk of having graft failure or GVHD, by administering aneffective amount of an anti-CD137 ADC described herein to the humanpatient at risk of having graft failure or GVHD, and subsequentlyadministering a transplant to the human subject. In some embodiments,the ADC is administered to the human patient as a single dose.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B graphically depict results of an in vitro cell bindingassay including a murine BBK2 (i.e., “BBK-mIgG1”) and a negative control(i.e., “mIgG1”) with Jurkat cells (i.e., a human T lymphocyte cell line)that over-express CD137.

FIGS. 2A and 2B graphically depict results of an in vitro T cell killingassay including an anti-CD137-amanitin ADC (i.e., “CD137-Amanitin”) andan anti-T cell specific-amanitin ADC (i.e., “anti-Tcell-Amanitin”) incomparison to a negative control (i.e., “hIgG-Amanitin”). The resultsshow the number of viable activated cells (FIG. 2A) or viablenon-activated cells (FIG. 2B) of each ADC (y-axis) as a function of ADCconcentration (x-axis).

FIG. 3 graphically depicts the results of an in vivo assay comparing thepercent survival of mice (y-axis) as a function of days post-transplant(x-axis) for an anti-CD137-amanitin ADC in comparison to controls PBS,an anti-CD137 antibody (naked), and isotype-amanitin in a NSG mousexeno-GVHD model.

FIG. 4 graphically depicts the results of an in vivo assay comparing thenumber of human T cells detected in the peripheral blood (y-axis) as afunction of days post-transplant (x-axis) for an anti-CD137-amanitin ADCin comparison to controls PBS, an anti-CD137 antibody (naked), andisotype-amanitin in a NSG mouse xeno-GVHD model.

FIGS. 5A and 5B graphically depict the results of in vivo assays fordetermining engraftment rates (FIG. 5A) and T cell frequency (FIG. 5B)in a humanized NSG-SGM3 mouse model where engraftment and T cellfrequency were measured at day 5, day 9, day 14, day 22 and at day 27post-transplant for an anti-CD137-amanitin ADC in comparison to controlsPBS, an anti-CD137-BBK antibody (naked), an isotype-amanitin, and ananti-Tcell-Amanitin ADC in a NSG mouse xeno-GVHD model.

DEFINITIONS

As used herein, the term “about” refers to a value that is within 10%above or below the value being described. For example, the term “about 5nM” indicates a range of from 4.5 nM to 5.5 nM.

As used herein, the term “allogeneic” refers to cells or tissues fromindividuals belonging to the same species but genetically different, andare therefore immunologically incompatible. Thus, the term “allogeneiccells” refers to cell types that are genetically distinct, yet belongingto the same species. Typically, the term “allogeneic” is used to definecells, such as stem cells, that are transplanted from a donor to arecipient of the same species.

As used herein, the term “amatoxin” refers to a member of the amatoxinfamily of peptides produced by Amanita phalloides mushrooms, or avariant or derivative thereof, such as a variant or derivative thereofcapable of inhibiting RNA polymerase II activity. Amatoxins useful inconjunction with the compositions and methods described herein includeα-amanitin, 3-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide,amanullin, amanullinic acid, and proamanullin, as well as derivativesthereof, such as a derivative described by any of formulas (I), (IA),and (II) described herein.

The term “antagonist” as used herein describes any molecule thatinhibits or reduces the biological activity of a target molecule, e.g.,CD137.

As used herein, the term “antibody” refers to an immunoglobulin moleculethat specifically binds to, or is immunologically reactive with, aparticular antigen, and includes polyclonal, monoclonal, geneticallyengineered, and otherwise modified forms of antibodies, including butnot limited to chimeric antibodies, humanized antibodies,heteroconjugate antibodies (e.g., bi-, tri-, and tetra-specificantibodies, diabodies, triabodies, and tetrabodies), and antigen bindingfragments of antibodies, including, for example, Fab′, F(ab′)₂, Fab, Fv,rIgG, and scFv fragments. Unless otherwise indicated, the term“monoclonal antibody” (mAb) is meant to include both intact molecules,as well as antibody fragments (including, for example, Fab and F(ab′)₂fragments) that are capable of specifically binding to a target protein.As used herein, the Fab and F(ab′)₂ fragments refer to antibodyfragments that lack the Fc fragment of an intact antibody. Examples ofthese antibody fragments are described herein.

Depending on the amino acid sequences of the constant domains of theheavy chains of antibodies, antibodies can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well knownand described generally in, for example, Abbas et al. Cellular and Mol.Immunology, 4th ed. (2000). An antibody may be part of a larger fusionmolecule, formed by covalent or non-covalent association of the antibodywith one or more other proteins or peptides.

The term “antigen-binding fragment,” as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to a target antigen. The antigen-binding function of an antibodycan be performed by fragments of a full-length antibody. The antibodyfragments can be, for example, a Fab, F(ab′)₂, scFv, diabody, atriabody, an affibody, a nanobody, an aptamer, or a domain antibody.Examples of binding fragments encompassed of the term “antigen-bindingfragment” of an antibody include, but are not limited to: (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L),and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H1) domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb including V_(H) and V_(L) domains; (vi) adAb fragment that consists of a V_(H) domain (see, e.g., Ward et al.,Nature 341:544-546, 1989); (vii) a dAb which consists of a V_(H) or aV_(L) domain; (viii) an isolated complementarity determining region(CDR); and (ix) a combination of two or more (e.g., two, three, four,five, or six) isolated CDRs which may optionally be joined by asynthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv (scFv). Theseantibody fragments can be obtained using conventional techniques knownto those of skill in the art, and the fragments can be screened forutility in the same manner as intact antibodies. Antigen-bindingfragments can be produced by recombinant DNA techniques, enzymatic orchemical cleavage of intact immunoglobulins, or, in certain cases, bychemical peptide synthesis procedures known in the art.

As used herein, the term “anti-CD137 antibody” refers to a protein orpeptide-containing molecule that includes at least a portion of animmunoglobulin molecule, such as but not limited to at least one CDR ofa heavy or light chain or a ligand binding portion thereof, a heavychain or light chain variable region, a heavy chain or light chainconstant region, a framework region, or any portion thereof, that iscapable of specifically binding to CD137. Anti-CD137 antibodies alsoinclude antibody-like protein scaffolds, such as the tenth fibronectintype III domain (¹⁰Fn3), which contains BC, DE, and FG structural loopssimilar in structure and solvent accessibility to antibody CDRs. Thetertiary structure of the ¹⁰Fn3 domain resembles that of the variableregion of the IgG heavy chain, and one of skill in the art can graft,for example, the CDRs of an anti-CD137 monoclonal antibody onto thefibronectin scaffold by replacing residues of the BC, DE, and FG loopsof ¹⁰Fn3 with residues from the CDRH-1, CDRH-2, or CDRH-3 regions of ananti-CD137 monoclonal antibody. In one embodiment, an anti-CD137antibody specifically binds to human CD137.

As used herein, the term “bispecific antibody” refers to, for example, amonoclonal, often a human or humanized antibody that is capable ofbinding at least two different antigens. For instance, one of thebinding specificities can be directed towards a T cell surface antigen,such as CD137, the other can be for a different T cell surface antigenor another cell surface protein, such as a receptor or receptor subunitinvolved in a signal transduction pathway that prohibits or limits cellgrowth, among others.

As used herein, the term “chimeric” antibody refers to refers toantibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (See, for example, U.S. Pat. No. 4,816,567and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855). Inone embodiment, a chimeric antibody comprises murine heavy and lightchain variable regions and human light and heavy chain constant regions.

As used herein, the terms “complementarity determining region” and “CDR”refer to a hypervariable region found both in the light chain and theheavy chain variable domains of an antibody. The more highly conservedportions of variable domains are referred to as framework regions (FRs).The amino acid positions that delineate a hypervariable region of anantibody can vary, depending on the context and the various definitionsknown in the art. Some positions within a variable domain may be viewedas hybrid hypervariable positions in that these positions can be deemedto be within a hypervariable region under one set of criteria whilebeing deemed to be outside a hypervariable region under a different setof criteria. One or more of these positions can also be found inextended hypervariable regions. The antibodies described herein maycontain modifications in these hybrid hypervariable positions. Thevariable domains of native heavy and light chains each comprise fourframework regions that primarily adopt a 3-sheet configuration,connected by three CDRs, which form loops that connect, and in somecases form part of, the 1-sheet structure. The CDRs in each chain areheld together in close proximity by the framework regions in the orderFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the otherantibody chains, contribute to the formation of the target binding siteof antibodies (e.g., see Kabat et al., Sequences of Proteins ofImmunological Interest, National Institute of Health, Bethesda, Md.,1987 or http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi).Numbering of immunoglobulin amino acid residues, including CDRs, can beperformed according to the immunoglobulin amino acid residue numberingsystem of Kabat et al.

As used herein, the term “conjugate” refers to a compound formed by thechemical bonding of a reactive functional group of one molecule, such asan antibody or antigen-binding fragment thereof, with an appropriatelyreactive functional group of another molecule, such as a cytotoxindescribed herein. Conjugates may include a linker between the twomolecules bound to one another, e.g., between an antibody and acytotoxin. Examples of linkers that can be used for the formation of aconjugate include peptide-containing linkers, such as those that containnaturally occurring or non-naturally occurring amino acids, such asD-amino acids. Linkers can be prepared using a variety of strategiesdescribed herein and known in the art. Depending on the reactivecomponents therein, a linker may be cleaved, for example, by enzymatichydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysisunder basic conditions, oxidation, disulfide reduction, nucleophiliccleavage, or organometallic cleavage (see, for example, Leriche et al.,Bioorg. Med. Chem., 20:571-582, 2012). Notably, the term “conjugate”(when referring to a compound) is also referred to interchangeablyherein as a “drug antibody conjugate” or an “antibody drug conjugate(ADC)”.

As used herein, the term “coupling reaction” refers to a chemicalreaction in which two or more substituents suitable for reaction withone another react so as to form a chemical moiety that joins (e.g.,covalently) the molecular fragments bound to each substituent. Couplingreactions include those in which a reactive substituent bound to afragment that is a cytotoxin, such as a cytotoxin known in the art ordescribed herein, reacts with a suitably reactive substituent bound to afragment that is an for CD137 known in the art or described herein.Examples of suitably reactive substituents include anucleophile/electrophile pair (e.g., a thiol/haloalkyl pair, anamine/carbonyl pair, or a thiol/α,β-unsaturated carbonyl pair, amongothers), a diene/dienophile pair (e.g., an azide/alkyne pair, amongothers), and the like. Coupling reactions include, without limitation,thiol alkylation, hydroxyl alkylation, amine alkylation, aminecondensation, amidation, esterification, disulfide formation,cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgencycloaddition, among others), nucleophilic aromatic substitution,electrophilic aromatic substitution, and other reactive modalities knownin the art or described herein.

As used herein, the term “donor” refers to a human or animal from whichone or more cells are isolated prior to administration of the cells, orprogeny thereof, into a recipient. The one or more cells may be, forexample, a population of hematopoietic stem cells.

As used herein, the term “diabody” refers to a bivalent antibodycontaining two polypeptide chains, in which each polypeptide chainincludes V_(H) and V_(L) domains joined by a linker that is too short(e.g., a linker composed of five amino acids) to allow forintramolecular association of V_(H) and V_(L) domains on the samepeptide chain. This configuration forces each domain to pair with acomplementary domain on another polypeptide chain so as to form ahomodimeric structure. Accordingly, the term “triabody” refers totrivalent antibodies comprising three peptide chains, each of whichcontains one V_(H) domain and one V_(L) domain joined by a linker thatis exceedingly short (e.g., a linker composed of 1-2 amino acids) topermit intramolecular association of V_(H) and V_(L) domains within thesame peptide chain. In order to fold into their native structures,peptides configured in this way typically trimerize so as to positionthe V_(H) and V_(L) domains of neighboring peptide chains spatiallyproximal to one another (see, for example, Holliger et al., Proc. Natl.Acad. Sci. USA 90:6444-48, 1993).

As used herein, a “dual variable domain immunoglobulin” (“DVD-Ig”)refers to an antibody that combines the target-binding variable domainsof two monoclonal antibodies via linkers to create a tetravalent,dual-targeting single agent (see, for example, Gu et al., Meth.Enzymol., 502:25-41, 2012).

As used herein, the term “endogenous” describes a substance, such as amolecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or acell of hematopoietic lineage, such as a megakaryocyte, thrombocyte,platelet, erythrocyte, mast cell, myoblast, basophil, neutrophil,eosinophil, microglial cell, granulocyte, monocyte, osteoclast,antigen-presenting cell, macrophage, dendritic cell, natural killercell, T cell, or B cell) that is found naturally in a particularorganism, such as a human patient.

As used herein, the term “exogenous” describes a substance, such as amolecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or acell of hematopoietic lineage, such as a megakaryocyte, thrombocyte,platelet, erythrocyte, mast cell, myoblast, basophil, neutrophil,eosinophil, microglial cell, granulocyte, monocyte, osteoclast,antigen-presenting cell, macrophage, dendritic cell, natural killercell, T cell, or B cell) that is not found naturally in a particularorganism, such as a human patient. Exogenous substances include thosethat are provided from an external source to an organism or to culturedmatter extracted therefrom.

As used herein, the term “framework region”, “FR”, or “FW region”includes amino acid residues that are adjacent to the CDRs within avariable region of an antibody, or antigen-binding fragment thereof. FWregion residues may be present in, for example, human antibodies,humanized antibodies, monoclonal antibodies, antibody fragments, Fabfragments, single chain antibody fragments, scFv fragments, antibodydomains, and bispecific antibodies, among others.

As used herein, the term “half-life” refers to the time it takes for theplasma concentration of the antibody drug in the body to be reduced byone half or 50%. This 50% reduction in serum concentration reflects theamount of drug circulating and not removed by the natural methods ofantibody clearance.

As used herein, the term “hematopoietic stem cells” (“HSCs”) refers toimmature blood cells having the capacity to self-renew and todifferentiate into mature blood cells comprising diverse lineagesincluding but not limited to granulocytes (e.g., promyelocytes,neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes,erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producingmegakaryocytes, platelets), monocytes (e.g., monocytes, macrophages),dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NKcells, B cells and T cells). Such cells may include CD34⁺ cells. CD34⁺cells are immature cells that express the CD34 cell surface marker. Inhumans, CD34+ cells are believed to include a subpopulation of cellswith the stem cell properties defined above, whereas in mice, HSCs areCD34-. In addition, HSCs also refer to long term repopulating HSCs(LT-HSC) and short term repopulating HSCs (ST-HSC). LT-HSCs and ST-HSCsare differentiated, based on functional potential and on cell surfacemarker expression. For example, human HSCs are CD34+, CD38−, CD45RA−,CD90+, CD49F+, and lin− (negative for mature lineage markers includingCD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A). Inmice, bone marrow LT-HSCs are CD34−, SCA-1+, C-kit+, CD135−,Slamfl/CD150+, CD48−, and lin− (negative for mature lineage markersincluding Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra), whereasST-HSCs are CD34+, SCA-1+, C-kit+, CD135−, Slamfl/CD150+, and lin−(negative for mature lineage markers including Ter119, CD11b, Gr1, CD3,CD4, CD8, B220, IL7ra). In addition, ST-HSCs are less quiescent and moreproliferative than LT-HSCs under homeostatic conditions. However, LT-HSChave greater self-renewal potential (i.e., they survive throughoutadulthood, and can be serially transplanted through successiverecipients), whereas ST-HSCs have limited self-renewal (i.e., theysurvive for only a limited period of time, and do not possess serialtransplantation potential). Any of these HSCs can be used in the methodsdescribed herein. ST-HSCs are particularly useful because they arehighly proliferative and thus, can more quickly give rise todifferentiated progeny.

As used herein, the term “hematopoietic stem cell functional potential”refers to the functional properties of hematopoietic stem cells whichinclude 1) multi-potency (which refers to the ability to differentiateinto multiple different blood lineages including, but not limited to,granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils),erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,megakaryoblasts, platelet producing megakaryocytes, platelets),monocytes (e.g., monocytes, macrophages), dendritic cells, microglia,osteoclasts, and lymphocytes (e.g., NK cells, T cells and B cells), 2)self-renewal (which refers to the ability of hematopoietic stem cells togive rise to daughter cells that have equivalent potential as the mothercell, and further that this ability can repeatedly occur throughout thelifetime of an individual without exhaustion), and 3) the ability ofhematopoietic stem cells or progeny thereof to be reintroduced into atransplant recipient whereupon they home to the hematopoietic stem cellniche and re-establish productive and sustained hematopoiesis.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (for example, all CDRs,framework regions, C_(L), C_(H) domains (e.g., C_(H1), C_(H)2, C_(H)3),hinge, and V_(L) and V_(H) domains) is substantially non-immunogenic inhumans, with only minor sequence changes or variations. A human antibodycan be produced in a human cell (for example, by recombinant expression)or by a non-human animal or a prokaryotic or eukaryotic cell that iscapable of expressing functionally rearranged human immunoglobulin (suchas heavy chain and/or light chain) genes. When a human antibody is asingle chain antibody, it can include a linker peptide that is not foundin native human antibodies. For example, an Fv can contain a linkerpeptide, such as two to about eight glycine or other amino acidresidues, which connects the variable region of the heavy chain and thevariable region of the light chain. Such linker peptides are consideredto be of human origin. Human antibodies can be made by a variety ofmethods known in the art including phage display methods using antibodylibraries derived from human immunoglobulin sequences. Human antibodiescan also be produced using transgenic mice that are incapable ofexpressing functional endogenous immunoglobulins, but which can expresshuman immunoglobulin genes.

A “humanized” antibody refers to a non-human antibody that containsminimal sequences derived from non-human immunoglobulin. Thus,“humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. In general, a humanized antibody contains substantially all ofat least one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin. All or substantially all of the FW regions may also bethose of a human immunoglobulin sequence. The humanized antibody canalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin consensus sequence.Methods of antibody humanization are known in the art.

In one embodiment, a humanized antibody is a human antibody (recipientantibody) in which residues from CDRs of the recipient are replaced byresidues from CDRs of a non-human species (donor antibody) such asmouse, rat, rabbit, or nonhuman primate having the desired specificity,affinity, and/or capacity. In some instances, framework region (FR)residues of the human antibody are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues thatare not found in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance. Ingeneral, a humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human antibody, and all or substantially all of the FRs are those ofa human immunoglobulin sequence. The humanized antibody optionally willalso comprise at least a portion of an antibody constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See alsothe following review articles and references cited therein: Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994).

The terms “full length antibody” and “intact antibody” are used hereininterchangeably to refer to an antibody in its substantially intactform, and not an antibody fragment as defined herein. Thus, for an IgGantibody, an intact antibody comprises two heavy chains each comprisinga variable region, a constant region and an Fc region, and two lightchains each comprising a variable region and a constant region. Morespecifically, an intact IgG comprises two light chains each comprising alight chain variable region (VL) and a light chain constant region (CL),and comprises two heavy chains each comprising a heavy chain variableregion (VH) and three heavy chain constant regions (CH1, CH2, and CH3).CH2 and CH3 represent the Fc region of the heavy chain.

As used herein, the term “microtubule-binding agent” refers to acompound which acts by disrupting the microtubular network that isessential for mitotic and interphase cellular function. Examples of amicrotubule-binding agent include, but are not limited to, maytasine,maytansinoids, and derivatives thereof, such as those described hereinor known in the art, vinca alkaloids, such as vinblastine, vinblastinesulfate, vincristine, vincristine sulfate, vindesine, and vinorelbine,taxanes, such as docetaxel and paclitaxel, macrolides, such asdiscodermolides, cochicine, and epothilones, and derivatives thereof,such as epothilone B or a derivative thereof. Paclitaxel is marketed asTAXOL®; docetaxel as TAXOTERE®; vinblastine sulfate as VINBLASTIN R.P®;and vincristine sulfate as FARMISTIN®. Also included are the genericforms of paclitaxel as well as various dosage forms of paclitaxel.Generic forms of paclitaxel include, but are not limited to, betaxololhydrochloride. Various dosage forms of paclitaxel include, but are notlimited to albumin nanoparticle paclitaxel marketed as ABRAXANE®;ONXOL®, CYTOTAX®. Discodermolide can be obtained, e.g., as disclosed inU.S. Pat. No. 5,010,099. Also included are epotholine derivatives whichare disclosed in U.S. Pat. No. 6,194,181, WO9810121, WO9825929,WO9808849, WO9943653, WO9822461 and WO0031247, the disclosures of eachof which are incorporated herein by reference.

As used herein, the term “monoclonal antibody” refers to an antibodythat is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

As used herein, the term “patient at risk for GVHD” refers to a patientwith one or more risk factors for developing GVHD. Risk factors include,but are not limited to, allogeneic donor transplant (e.g.,transplantation of hematopoietic stem cells from a bone marrowtransplant), including mismatched human leucocyte antigen (HLA) donorand sex mismatched donor, T cell replete stem cell transplant, donor andrecipient age, presence of cytomegalovirus (CMV) or CMV antibodies intransplant donor or host, increased dose of total-body irradiation(TBI), conditioning regimen intensity, acute GVHD prophylaxis, lack ofprotective environments, splenectomy, immunoglobulin use, underlyingdisease, ABO compatibility, prior exposure to herpes viruses, donorblood transfusions, performance score, antibiotic gut decontamination,and post-allogeneic transplant blood transfusions.

As used herein, the term “patient at risk for an autoimmune disease”refers to a patient with one or more risk factors for developing anautoimmune disease. Risk factors include, but are not limited to, age(young to middle aged), sex (female), ethnicity (African American,American Indian, or Latino), family history of autoimmune diseases,exposure to environmental agents, previous infection, chronicinflammation, and donor transplantation (e.g., transplantation ofhematopoietic stem cells from a bone marrow transplant). As used herein,the term “recipient” refers to a patient that receives a transplant,such as a transplant containing a population of hematopoietic stemcells. The transplanted cells administered to a recipient may be, e.g.,autologous, syngeneic, or allogeneic cells.

As used herein, the term “sample” refers to a specimen (e.g., blood,blood component (e.g., serum or plasma), urine, saliva, amniotic fluid,cerebrospinal fluid, tissue (e.g., placental or dermal), pancreaticfluid, chorionic villus sample, and cells) taken from a subject.

As used herein, the term “scFv” refers to a single chain Fv antibody inwhich the variable domains of the heavy chain and the light chain froman antibody have been joined to form one chain. scFv fragments contain asingle polypeptide chain that includes the variable region of anantibody light chain (V_(L)) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) andthe variable region of an antibody heavy chain (V_(H)) (e.g., CDR-H1,CDR-H2, and/or CDR-H3) separated by a linker. The linker that joins theV_(L) and V_(H) regions of a scFv fragment can be a peptide linkercomposed of proteinogenic amino acids. Alternative linkers can be usedto so as to increase the resistance of the scFv fragment to proteolyticdegradation (for example, linkers containing D-amino acids), in order toenhance the solubility of the scFv fragment (for example, hydrophiliclinkers such as polyethylene glycol-containing linkers or polypeptidescontaining repeating glycine and serine residues), to improve thebiophysical stability of the molecule (for example, a linker containingcysteine residues that form intramolecular or intermolecular disulfidebonds), or to attenuate the immunogenicity of the scFv fragment (forexample, linkers containing glycosylation sites). It will also beunderstood by one of ordinary skill in the art that the variable regionsof the scFv molecules described herein can be modified such that theyvary in amino acid sequence from the antibody molecule from which theywere derived. For example, nucleotide or amino acid substitutionsleading to conservative substitutions or changes at amino acid residuescan be made (e.g., in CDR and/or framework residues) so as to preserveor enhance the ability of the scFv to bind to the antigen recognized bythe corresponding antibody.

The terms “specific binding” or “specifically binding”, as used herein,refers to the ability of an antibody (or an ADC) to recognize and bindto a specific protein structure (epitope) rather than to proteinsgenerally. If an antibody or ADC is specific for epitope “A”, thepresence of a molecule containing epitope A (or free, unlabeled A), in areaction containing labeled “A” and the antibody, will reduce the amountof labeled A bound to the antibody or ADC. By way of example, anantibody “binds specifically” to a target if the antibody, when labeled,can be competed away from its target by the corresponding non-labeledantibody. In one embodiment, an antibody specifically binds to a target,e.g., CD137, if the antibody has a K_(D) for the target of at leastabout 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M,10⁻¹² M, or less (less meaning a number that is less than 10⁻¹², e.g.10⁻¹³). In one embodiment, the term “specific binding to CD137” or“specifically binds to CD137,” as used herein, refers to an antibody oran ADC that binds to CD137 and has a dissociation constant (K_(D)) of1.0×10⁻⁷ M or less, as determined by surface plasmon resonance. In oneembodiment, K_(D) is determined according to standard bio-layerinterferometery (BLI). It shall be understood, however, that theantibody or ADC may be capable of specifically binding to two or moreantigens which are related in sequence. For example, in one embodiment,an antibody can specifically bind to both human and a non-human (e.g.,mouse or non-human primate) orthologs of CD137.

As used herein, the terms “subject” and “patient” refer to an organism,such as a human, that receives treatment for a particular disease orcondition as described herein. For instance, a patient, such as a humanpatient, may receive treatment prior to hematopoietic stem celltransplant therapy in order to treat or prevent GVHD by administrationof an antibody, antigen-binding fragment thereof, or ligand as describedherein capable of binding CD137.

As used herein, the phrase “substantially cleared from the blood” refersto a point in time following administration of a therapeutic agent (suchas an anti-CD137 antibody, antigen-binding fragment thereof, ADC, orsoluble ligand) to a patient when the concentration of the therapeuticagent in a blood sample isolated from the patient is such that thetherapeutic agent is not detectable by conventional means (for instance,such that the therapeutic agent is not detectable above the noisethreshold of the device or assay used to detect the therapeutic agent).A variety of techniques known in the art can be used to detectantibodies, antibody fragments and protein ligands, such as ELISA-baseddetection assays known in the art or described herein. Additional assaysthat can be used to detect antibodies, antibody fragments, and proteinligands include immunoprecipitation techniques and immunoblot assays,among others known in the art.

As used herein, the phrase “stem cell disorder” broadly refers to anydisease, disorder, or condition that may be treated or cured byconditioning a subject's target tissues, and/or by ablating anendogenous stem cell population in a target tissue (e.g., ablating anendogenous hematopoietic stem or progenitor cell population from asubject's bone marrow tissue) and/or by engrafting or transplanting stemcells in a subject's target tissues. For example, Type I diabetes hasbeen shown to be cured by hematopoietic stem cell transplant and maybenefit from conditioning in accordance with the compositions andmethods described herein. Additional disorders that can be treated usingthe compositions and methods described herein include, withoutlimitation, sickle cell anemia, thalassemias, Fanconi anemia,Wiskott-Aldrich syndrome, ADA SCID, HIV/AIDS, metachromaticleukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamondsyndrome. The subject may have or be affected by an inherited blooddisorder (e.g., sickle cell anemia) or an autoimmune disorder.Additionally or alternatively, the subject may have or be affected by amalignancy, such as a malignancy selected from the group consisting ofhematologic cancers (e.g., leukemia, lymphoma, multiple myeloma, ormyelodysplastic syndrome) and neuroblastoma. In some embodiments, thesubject has or is otherwise affected by a metabolic disorder. Forexample, the subject may suffer or otherwise be affected by a metabolicdisorder selected from the group consisting of glycogen storagediseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,sphingolipidoses, metachromatic leukodystrophy, or any other diseases ordisorders which may benefit from the treatments and therapies disclosedherein and including, without limitation, severe combinedimmunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM)syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis,osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemiamajor, sickle cell disease, systemic sclerosis, systemic lupuserythematosus, multiple sclerosis, juvenile rheumatoid arthritis andthose diseases, or disorders described in “Bone Marrow Transplantationfor Non-Malignant Disease,” ASH Education Book, 1:319-338 (2000), thedisclosure of which is incorporated herein by reference in its entiretyas it pertains to pathologies that may be treated by administration ofhematopoietic stem cell transplant therapy.

As used herein, the term “suffering from disease” refers to a subject(e.g., a human) that is experiencing GVHD or an autoimmune disease. Itis not intended that the present invention be limited to any particularsigns or symptoms, nor disease. Thus, it is intended that the presentinvention encompass subjects that are experiencing any range of disease,from sub-clinical to full-blown disease, wherein the subject exhibits atleast some of the indicia (e.g., signs and symptoms) associated withGVHD or an autoimmune disease.

As used herein, the term “transfection” refers to any of a wide varietyof techniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, such as electroporation,lipofection, calcium-phosphate precipitation, DEAE-dextran transfectionand the like.

As used herein, the term “transplant” refers to any organ, body tissue,or cell(s) that has been transferred from its site of origin to arecipient site, or the act of doing so.

As used herein, the terms “treat” or “treatment” refer to therapeutictreatment, in which the object is to prevent or slow down (lessen) anundesired physiological change or disorder or to promote a beneficialphenotype in the patient being treated. Beneficial or desired clinicalresults include, but are not limited to, a decrease in the cell count orrelative concentration of CD137 positive cells, a decrease in thecellular and clinical manifestations of GVHD or an autoimmune disease,promoting the engraftment of exogenous hematopoietic cells in a patientas described herein and subsequent hematopoietic stem cell transplanttherapy. Additional beneficial results include an increase in the cellcount or relative concentration of hematopoietic stem cells sufferingfrom or at risk for GVHD. Beneficial results of therapy described hereinmay also include an increase in the cell count or relative concentrationof one or more cells of hematopoietic lineage, such as a megakaryocyte,thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil,neutrophil, eosinophil, microglial cell, granulocyte, monocyte,osteoclast, antigen-presenting cell, macrophage, dendritic cell, naturalkiller cell, T cell, or B cell, following hematopoietic stem celltransplant therapy.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to an amount that is sufficient to achieve thedesired result or to have an effect on GVHD or an autoimmune disease.The specific therapeutically effective dose level for any particularpatient will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration; the route of administration; therate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed and like factors well known in the art. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of a heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites (CDRs).

As used herein, the term “vector” includes a nucleic acid vector, suchas a plasmid, a DNA vector, a plasmid, a RNA vector, virus, or othersuitable replicon. Expression vectors described herein may contain apolynucleotide sequence as well as, for example, additional sequenceelements used for the expression of proteins and/or the integration ofthese polynucleotide sequences into the genome of a mammalian cell.Certain vectors that can be used for the expression of antibodies andantibody fragments of the invention include plasmids that containregulatory sequences, such as promoter and enhancer regions, whichdirect gene transcription. Other useful vectors for expression ofantibodies and antibody fragments contain polynucleotide sequences thatenhance the rate of translation of these genes or improve the stabilityor nuclear export of the mRNA that results from gene transcription.These sequence elements may include, for example, 5′ and 3′ untranslatedregions and a polyadenylation signal site in order to direct efficienttranscription of the gene carried on the expression vector. Theexpression vectors described herein may also contain a polynucleotideencoding a marker for selection of cells that contain such a vector.Examples of a suitable marker include genes that encode resistance toantibiotics, such as ampicillin, chloramphenicol, kanamycin, andnourseothricin.

As used herein, the term “alkyl” refers to a straight- or branched-chainalkyl group having, for example, from 1 to 20 carbon atoms in the chain.Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl,hexyl, isohexyl, and the like.

As used herein, the term “alkylene” refers to a straight- orbranched-chain divalent alkyl group. The divalent positions may be onthe same or different atoms within the alkyl chain. Examples of alkyleneinclude methylene, ethylene, propylene, isopropylene, and the like.

As used herein, the term “heteroalkyl” refers to a straight orbranched-chain alkyl group having, for example, from 1 to 20 carbonatoms in the chain, and further containing one or more heteroatoms(e.g., oxygen, nitrogen, or sulfur, among others) in the chain.

As used herein, the term “heteroalkylene” refers to a straight- orbranched-chain divalent heteroalkyl group. The divalent positions may beon the same or different atoms within the heteroalkyl chain.

As used herein, the term “alkenyl” refers to a straight- orbranched-chain alkenyl group having, for example, from 2 to 20 carbonatoms in the chain. Examples of alkenyl groups include vinyl, propenyl,isopropenyl, butenyl, tert-butylenyl, hexenyl, and the like.

As used herein, the term “alkenylene” refers to a straight- orbranched-chain divalent alkenyl group. The divalent positions may be onthe same or different atoms within the alkenyl chain. Examples ofalkenylene include ethenylene, propenylene, isopropenylene, butenylene,and the like.

As used herein, the term “heteroalkenyl” refers to a straight- orbranched-chain alkenyl group having, for example, from 2 to 20 carbonatoms in the chain, and further containing one or more heteroatoms(e.g., oxygen, nitrogen, or sulfur, among others) in the chain.

As used herein, the term “heteroalkenylene” refers to a straight- orbranched-chain divalent heteroalkenyl group. The divalent positions maybe on the same or different atoms within the heteroalkenyl chain.

As used herein, the term “alkynyl” refers to a straight- orbranched-chain alkynyl group having, for example, from 2 to 20 carbonatoms in the chain. Examples of alkynyl groups include propargyl,butynyl, pentynyl, hexynyl, and the like.

As used herein, the term “alkynylene” refers to a straight- orbranched-chain divalent alkynyl group. The divalent positions may be onthe same or different atoms within the alkynyl chain.

As used herein, the term “heteroalkynyl” refers to a straight- orbranched-chain alkynyl group having, for example, from 2 to 20 carbonatoms in the chain, and further containing one or more heteroatoms(e.g., oxygen, nitrogen, or sulfur, among others) in the chain.

As used herein, the term “heteroalkynylene” refers to a straight- orbranched-chain divalent heteroalkynyl group. The divalent positions maybe on the same or different atoms within the heteroalkynyl chain.

As used herein, the term “cycloalkyl” refers to a monocyclic, or fused,bridged, or spiro polycyclic ring structure that is saturated and has,for example, from 3 to 12 carbon ring atoms. Examples of cycloalkylgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, and the like.

As used herein, the term “cycloalkylene” refers to a divalent cycloalkylgroup. The divalent positions may be on the same or different atomswithin the ring structure. Examples of cycloalkylene includecyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and thelike.

As used herein, the term “heterocyloalkyl” refers to a monocyclic, orfused, bridged, or spiro polycyclic ring structure that is saturated andhas, for example, from 3 to 12 ring atoms per ring structure selectedfrom carbon atoms and heteroatoms selected from, e.g., nitrogen, oxygen,and sulfur, among others. The ring structure may contain, for example,one or more oxo groups on carbon, nitrogen, or sulfur ring members.

As used herein, the term “heterocycloalkylene” refers to a divalentheterocyclolalkyl group. The divalent positions may be on the same ordifferent atoms within the ring structure. As used herein, the term“aryl” refers to a monocyclic or multicyclic aromatic ring systemcontaining, for example, from 6 to 19 carbon atoms. Aryl groups include,but are not limited to, phenyl, fluorenyl, naphthyl, and the like.

As used herein, the term “arylene” refers to a divalent aryl group. Thedivalent positions may be on the same or different atoms.

As used herein, the term “heteroaryl” refers to a monocyclicheteroaromatic, or a bicyclic or a tricyclic fused-ring heteroaromaticgroup. Heteroaryl groups include pyridyl, pyrrolyl, furyl, thienyl,imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadia-zolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl,benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl,benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl,benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl,quinolizinyl, quinazolinyl, pthalazinyl, quinoxalinyl, cinnolinyl,napthyridinyl, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl,pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl,5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl,pteridinyl, carbazolyl, xanthenyl, benzoquinolyl, and the like.

As used herein, the term “heteroarylene” refers to a divalent heteroarylgroup. The divalent positions may be on the same or different atoms.

Unless otherwise constrained by the definition of the individualsubstituent, the foregoing chemical moieties, such as “alkyl”,“alkylene”, “heteroalkyl”, “heteroalkylene”, “alkenyl”, “alkenylene”,“heteroalkenyl”, “heteroalkenylene”, “alkynyl”, “alkynylene”,“heteroalkynyl”, “heteroalkynylene”, “cycloalkyl”, “cycloalkylene”,“heterocyclolalkyl”, heterocycloalkylene”, “aryl,” “arylene”,“heteroaryl”, and “heteroarylene” groups can optionally be substitutedwith, for example, from 1 to 5 substituents selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,alkyl aryl, alkyl heteroaryl, alkyl cycloalkyl, alkyl heterocycloalkyl,amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl,alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl,alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy,mercapto, nitro, and the like. The substitution may include situationsin which neighboring substituents have undergone ring closure, such asring closure of vicinal functional substituents, to form, for instance,lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals,aminals, and hemiaminals, formed by ring closure, for example, tofurnish a protecting group.

DETAILED DESCRIPTION

The invention provides methods of preventing and treatinggraft-vs-host-disease (GVHD) and autoimmune diseases by administrationof an antibody, antigen-binding fragment thereof, ADC, or soluble ligandcapable of binding an antigen expressed by hematopoietic cells. Thisadministration can cause the selective depletion of a population ofexogenous T cells that are reactive against the host. The invention isbased in part on the discovery that an antibody, antigen-bindingfragment thereof, ADC, or soluble ligand, capable of binding CD137 canbe administered to a patient in in order to prevent and treat GVHD andautoimmune diseases, such as those arising from hematopoietic stem celltransplant therapy.

Prevention and treatment of GVHD, due to the administration ofanti-CD137 antibodies, antigen-binding fragments thereof, ADCs, orsoluble ligands can manifest in a variety of clinical symptoms (see,e.g., McDonald, Blood. 127:1544-1440, 2016, and Flowers et al., Blood.125:606-615, the disclosure of which is incorporated herein by referenceas it pertains, but is not limited to, the measureable clinical featuresof acute and chronic GVHD, respectively). Prevention and treatment ofGVHD and autoimmune diseases, due to the administration of anti-CD137antibodies, antigen-binding fragments thereof, or ADCs, can manifest ina variety of empirical measurements. For instance, depletion of CD137+positive cells can be determined by fluorescence activated cell sorting(FACS) analysis methods known in the art to measure CD137+ white bloodcell counts in peripheral blood during a post-transplant period, and/orby measuring recovery of marrow cells by donor cells in a bone marrowaspirate sample. Enumeration of the interferon-γ (IFN-γ)-producing Tcells in the peripheral blood of recipients can assess the efficacy ofanti-CD137 against GVHD and autoimmune diseases. The alteration ofimmune cell populations, as determined by FACS, can be indicative ofGVHD or an autoimmune disease. Finally, genetic and proteomic biomarkerstaken from the patient can also indicate GVHD or an autoimmune disease.

The sections that follow provide a description of antibodies,antigen-binding fragments thereof, ADCs, or soluble ligands that can beadministered to a patient suffering from or at risk for GVHD or anautoimmune disease as well as methods of administering such therapeuticsto the patient.

Anti-CD137 Antibodies and Ligands

The present invention is based in part on the discovery that antibodies,antigen-binding fragments thereof, and soluble ligands capable ofbinding CD137 (also referred to as CDw137, TNFRSF9, 4-1BB, and ILA) canbe used as therapeutic agents to prevent and treat GVHD fromhematopoietic stem cells in a patient suffering from or at risk for GVHDor an autoimmune disease. Additionally, it has been discovered thatligands that bind CD137, such as human CD137L, can be used as atherapeutic agent to prevent or treat patient suffering from or at riskfor GVHD. These ligands, such as soluble human CD137, can be covalentlybound to an effector domain, such as an Fc domain, for instance, inorder to promote antibody-dependent cell-mediated cytotoxicity (ADCC).

T cells have been shown to express CD137, as this antigen is atransmembrane TNF receptor superfamily of costimulatory molecules and isexpressed on a variety of hematopoietic cells and promotes T cellactivation and regulates proliferation and survival of T cells (see,e.g., Cannons et al., J. Immunol. 167:1313-1324, 2001, the disclosure ofwhich is incorporated herein by reference as it pertains to theexpression of CD137 by T cells). Antibodies, antigen-binding fragmentsthereof, and ligands can be identified using techniques known in the artand described herein, such as by immunization, computational modelingtechniques, and in vitro selection methods, such as the phage displayand cell-based display platforms described below.

Anti-CD137 antibodies that can be used to prevent and treat GVHD or anautoimmune disease by the methods disclosed herein include those thathave one or more, or all, of the following CDRs:

-   -   a. a CDR-H1 having the amino acid sequence STYWIS (SEQ ID NO:        1);    -   b. a CDR-H2 having the amino acid sequence KIYPGDSYTNYSPSFQG        (SEQ ID NO: 2);    -   c. a CDR-H3 having the amino acid sequence RGYGIFDY (SEQ ID NO:        3);    -   d. a CDR-L1 having the amino acid sequence SGDNIGDQYAH (SEQ ID        NO: 4)    -   e. a CDR-L2 having the amino acid sequence QDKNRPS (SEQ ID NO:        5); and    -   f. a CDR-L3 having the amino acid sequence ATYTGFGSLAV (SEQ ID        NO: 6)

Additional anti-CD137 antibodies that can be used to prevent and treatGVHD and autoimmune diseases by the methods disclosed herein includethose that have one or more, or all, of the following CDRs:

-   -   a. a CDR-H1 having the amino acid sequence STYWIS (SEQ ID NO:        1);    -   b. a CDR-H2 having the amino acid sequence KIYPGDSYTNYSPSFQG        (SEQ ID NO: 2);    -   c. a CDR-H3 having the amino acid sequence RGYGIFDY (SEQ ID NO:        3);    -   d. a CDR-L1 having the amino acid sequence SGDNIGDQYAH (SEQ ID        NO: 4)    -   e. a CDR-L2 having the amino acid sequence QDKNRPS (SEQ ID NO:        5); and    -   f. a CDR-L3 having the amino acid sequence STYTFVGFTTV (SEQ ID        NO: 7)

Additional anti-CD137 antibodies include those that have one or more, orall, of the following CDRs:

-   -   a. a CDR-H1 having the amino acid sequence NSYAIS (SEQ ID NO:        8);    -   b. a CDR-H2 having the amino acid sequence GIIPGFGTANYAQKFQG        (SEQ ID NO: 9);    -   c. a CDR-H3 having the amino acid sequence RKNEEDGGFDH (SEQ ID        NO: 10);    -   d. a CDR-L1 having the amino acid sequence SGDNLGDYYAS (SEQ ID        NO: 11)    -   e. a CDR-L2 having the amino acid sequence DDSNRPS (SEQ ID NO:        12); and    -   f. a CDR-L3 having the amino acid sequence QTWDGTLHFV (SEQ ID        NO: 13)

Additional anti-CD137 antibodies or ADCs include those that have one ormore, or all, of the following CDRs:

-   -   a. a CDR-H1 having the amino acid sequence SDYYMH (SEQ ID NO:        14);    -   b. a CDR-H2 having the amino acid sequence VISGSGSNTYYADSVKG        (SEQ ID NO: 15);    -   c. a CDR-H3 having the amino acid sequence RLYAQFEGDF (SEQ ID        NO: 16);    -   d. a CDR-L1 having the amino acid sequence SGDNIGSKYVS (SEQ ID        NO: 17)    -   e. a CDR-L2 having the amino acid sequence SDSERPS (SEQ ID NO:        18); and    -   f. a CDR-L3 having the amino acid sequence QSWDGSISRV (SEQ ID        NO: 19)

The foregoing antibodies are described, e.g., in U.S. Pat. No.9,468,678, the disclosure of which is incorporated herein by referenceas it pertains to anti-CD137 antibodies and antigen-binding fragmentsthereof. The antibodies and fragments thereof disclosed in U.S. Pat. No.9,468,678 can be used in conjunction with the methods disclosed herein.

In another embodiment, an anti-CD137 antibody that may be used in themethods and compositions (including ADCs) described herein is the murineanti-CD137 antibody BBK2 (Thermo Fisher; MS621 PABX) or an anti-CD137antibody comprising antigen binding regions corresponding to the BBK2antibody. The BBK2 antibody (which may also be referred to as a BBK-2antibody or an anti-4-1BB antibody), is a mouse monoclonal antibody(IgG1, kappa) that binds to the ectodomain of human 4-1BB recombinantprotein (4-1BB is also known as CD137). In certain embodiments, themethods and compositions of the disclosure include an anti-CD137antibody comprising the binding regions (e.g., the CDRs) of the BBK2antibody. In another embodiment, the methods and compositions of thedisclosure comprise an antibody that competitively inhibits the bindingof the BBK2 antibody to its epitope on CD137. In certain embodiments,the anti-CD137 antibody is humanized BBK2 or chimeric BBK2.

In one embodiment, the methods and compositions described herein includea chimeric anti-CD137 (ch-BBK2) antibody comprising the variable heavyand light chain regions of BBK2. In certain embodiments, the chimericBBK2 antibody is an IgG1 antibody comprising human constant regions. Theheavy chain amino acid sequence of ch-BBK2 is described in SEQ ID NO:23, and the light chain amino acid sequence of ch-BBK2 is described inSEQ ID NO: 24. The CDR regions (CDR1, CDR2, and CDR3) of each of theheavy and light chain sequences are described in bold below. Thevariable regions are italicized.

(SEQ ID NO: 23) QVQLQQPGAELVRPGASVKLSCKA SGYTFTSYW INWVKQRPGQGLEWIG NIYPSDSYT NYNQKFKDKATLTVDKSSNTVYMQLNSPTSEDSAVYYC TRNG VEGYPHYYAMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 24)DIQMTQTTSALSASLGDRVTIGCRA SQDLSNH LYWYQQKPDGTVKLLI YY TSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDVATYF CQQGYTLPY TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

The foregoing CDR regions (and BBK2 antibody) are described in Lee etal. (2002) European J of Immunogenetics 29(5):449-452. Thus, in oneembodiment, the VH CDR amino acid sequences of anti-CD137 antibody BBK2(including ch-BBK2) are as follows: SGYTFTSYW (VH CDR1; SEQ ID NO: 33);NIYPSDSYT (VH CDR2; SEQ ID NO: 34) and TRNGVEGYPHYYAME (VH CDR3; SEQ IDNO: 35). The VL CDR amino acid sequences of anti-CD137 antibody BBK2(including ch-BBK2) are as follows: SQDLSNH (VL CDR1; SEQ ID NO: 36);YYTS (VL CDR2; SEQ ID NO: 37) and CQQGYTLPY (VL CDR3; SEQ ID NO: 38).

Alternatively, the CDR regions of BBK2 can be defined according to Kabatnumbering. CDRs as defined by Kabat numbering are described below foreach of the heavy and light chain sequences (described in bold below).The variable regions of BBK2 are italicized.

(ch-BBK2 heavy chain; SEQ ID NO: 23) QVQLQQPGAELVRPGASVKLSCKASGYTFT

WVKQRPGQGLEWI G

KATLTVDKSSNTVYMQLNSPTSEDSAVYYC TR

WGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK(ch-BBK2 light chain; SEQ ID NO: 24) DIQMTQTTSALSASLGDRVTIGC

WYQQKPDGTVKLLIY

GVPSRFSGSGSGTDYSLTIRNLEQEDVATYFC

FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVQLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECThus, in one embodiment, the VH CDR amino acid sequences of anti-CD137antibody BBK2 (including ch-BBK2) are as follows: SYWIN (VH CDR1; SEQ IDNO: 25); NIYPSDSYTNYNQKFKD (VH CDR2; SEQ ID NO: 26) and NGVEGYPHYYAMEY(VH CDR3; SEQ ID NO: 27), and the VL CDR amino acid sequences ofanti-CD137 antibody BBK2 (including ch-BBK2) are as follows: RASQDLSNHLY(VL CDR1; SEQ ID NO: 29); YTSRLHS (VL CDR2; SEQ ID NO: 30) and QQGYTLPYT(VL CDR3; SEQ ID NO: 31).

The heavy chain variable region of BBK2 is set forth in SEQ ID NO: 28 asQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSNTVYMQLNSPTSEDSAVYYCTRNGVEGYPHYYAMEYWGQGTSVTVSS. The lightchain variable region of BBK2 is set forth in SEQ ID NO: 32 asDIQMTQTTSALSASLGDRVTIGCRASQDLSNHLYWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDVATYFCQQGYTLPYTFGGGTKLEIK. Anti-CD137 antibodies(including anti-CD137 ADCs) can comprise the heavy and light chainvariable region amino acid sequences as set forth in SEQ ID Nos: 28 and32, respectively.

In one embodiment, the anti-CD137 antibody, e.g., a chimeric (ch-BBK2)antibody or a humanized BBK2 antibody, comprises a heavy chain variableregion comprising a CDR1 comprising the amino acid sequence of SEQ IDNO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 26, anda CDR3 comprising the amino acid sequence of SEQ ID NO: 27; andcomprises a light chain variable region comprising a CDR1 comprising theamino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acidsequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequenceof SEQ ID NO: 31.

In one embodiment, the anti-CD137 antibody, e.g., a chimeric (ch-BBK2)antibody or a humanized BBK2 antibody, comprises a heavy chain variableregion comprising a CDR1 comprising the amino acid sequence of SEQ IDNO: 33, a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, anda CDR3 comprising the amino acid sequence of SEQ ID NO: 35; andcomprises a light chain variable region comprising a CDR1 comprising theamino acid sequence of SEQ ID NO: 36, a CDR2 comprising the amino acidsequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequenceof SEQ ID NO: 38.

Thus, BBK2, humanized BBK2, or chimeric BBK2 antibodies can be used inthe anti-CD137 ADCs and methods described herein. Each of theseantibodies can be conjugated to any of the cytotoxin described belowusing methods known in the art and those described herein.

Other anti-CD137 antibodies that can be used in conjunction with acytotoxin described herein can be identified using techniques known inthe art (e.g., hybridoma production). Hybridomas can be prepared using amurine system. Protocols for immunization and subsequent isolation ofsplenocytes for fusion are known in the art. Fusion partners andprocedures for hybridoma generation are also known. Human anti-CD137antibodies can also be generated in the HuMAb-Mouse® or XenoMouse™. Inmaking anti-CD137 antibodies, the CD137 antigen is isolated and/orpurified. The CD137 antigen may be a fragment of CD137 from theextracellular domain of CD137. Immunization of animals can be performedby any method known in the art. See, e.g., Harlow and Lane, Antibodies:A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methodsfor immunizing animals such as mice, rats, sheep, goats, pigs, cattleand horses are well known in the art. See, e.g., Harlow and Lane, supra,and U.S. Pat. No. 5,994,619. The CD137 antigen may be administered withan adjuvant to stimulate the immune response. Adjuvants known in the artinclude complete or incomplete Freund's adjuvant, RIBI (muramyldipeptides) or ISCOM (immunostimulating complexes). After immunizationof an animal with a CD137 antigen, antibody-producing immortalized celllines are prepared from cells isolated from the immunized animal. Afterimmunization, the animal is sacrificed and lymph node and/or splenic Bcells are immortalized by methods known in the art (e.g., oncogenetransfer, oncogenic virus transduction, exposure to carcinogenic ormutating compounds, fusion with an immortalized cell, e.g., a myelomacell, and inactivating a tumor suppressor gene. See, e.g., Harlow andLane, supra. Hybridomas can be selected, cloned and further screened fordesirable characteristics, including robust growth, high antibodyproduction and desirable antibody characteristics.

Anti-CD137 antibodies can be generated from an isolated nucleic acidmolecule that comprises a nucleotide sequence encoding an amino acidsequence of a CD137 binding molecule provided by the present disclosure.The amino acid sequence encoded by the nucleotide sequence may be anyportion of an antibody, such as a CDR, a sequence comprising one, two,or three CDRs, a variable region of a heavy chain, variable region of alight chain, or may be a full-length heavy chain or full length lightchain. A nucleic acid of the disclosure can be, for example, DNA or RNA,and may or may not contain intronic sequences. Typically, the nucleicacid is a cDNA molecule.

In addition to antibodies, and antigen-binding fragments, soluble CD137ligands, such as human CD137 ligand, can be administered to a patientaccording to the methods described herein to condition a patient priorto hematopoietic stem cell transplant therapy. For instance, CD137ligands, such as human CD137 ligand, can be conjugated to a cytotoxin(e.g., according to the methods described below or known in the art) oranother effector molecule, such as an Fc domain. Maytansine cytotoxinsfor use with the methods described herein include, for example, humanCD137 ligand-IgG1 Fc conjugates, human CD137 ligand-IgG2 Fc conjugates,human CD137 ligand-IgG3 Fc conjugates, human CD137 ligand-IgG4 Fcconjugates, human CD137 ligand-IgA Fc conjugates, human CD137 ligand-IgEFc conjugates, human CD137 ligand-IgM Fc conjugates, and human CD137ligand-IgD Fc conjugates.

Antibodies and ligands for use in conjunction with the compositions andmethods described herein include variants of those antibodies describedabove, such as antibody fragments that contain or lack an Fc domain, aswell as humanized variants of non-human antibodies described herein andantibody-like protein scaffolds (e.g., ¹⁰Fn3 domains) containing one ormore, or all, of the CDRs or equivalent regions thereof of an antibody,antibody fragment, or soluble ligand described herein.

Methods of Identifying Antibodies and Ligands

Methods for high throughput screening of libraries of antibodies,antibody fragments, and ligands for molecules capable of binding CD137can be used to identify and affinity mature agents that are, forexample, useful for preventing and treating GVHD or autoimmune diseases.Such methods include in vitro display techniques known in the art, suchas phage display, bacterial display, yeast display, mammalian celldisplay, ribosome display, mRNA display, and cDNA display, among others.The use of phage display to isolate antibodies, antigen-bindingfragments, or ligands that bind biologically relevant molecules has beenreviewed, for example, in Felici et al., Biotechnol. Annual Rev.1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45,1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998, thedisclosures of each of which are incorporated herein by reference asthey pertain to in vitro display techniques. Randomized combinatorialpeptide libraries have been constructed to select for polypeptides thatbind cell surface antigens as described in Kay, Perspect. Drug DiscoveryDes. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996, thedisclosures of each of which are incorporated herein by reference asthey pertain to the discovery of antigen-binding molecules. Proteins,such as multimeric proteins, have been successfully phage-displayed asfunctional molecules (see, for example, EP 0349578; EP 4527839; and EP0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-841992, the disclosures of each of which are incorporated herein byreference as they pertain to the use of in vitro display techniques forthe discovery of antigen-binding molecules. In addition, functionalantibody fragments, such as Fab and scFv fragments, have been expressedin in vitro display formats (see, for example, McCafferty et al., Nature348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, thedisclosures of each of which are incorporated herein by reference asthey pertain to in vitro display platforms for the discovery ofantigen-binding molecules). Human anti-CD137 antibodies can also begenerated, for example, in the HuMAb-Mouse® or XenoMouse™. Thesetechniques, among others, can be used to identify and improve theaffinity of antibodies, antibody fragments, and ligands that bind CD137that can in turn be used to deplete hematopoietic cells in a patient.

In addition to in vitro display techniques, computational modelingtechniques can be used to design and identify anti-CD137 antibodies,antibody fragments and ligands in silico, for instance, using theprocedures described in US 2013/0288373, the disclosure of which isincorporated herein as it pertains to molecular modeling methods foridentifying anti-CD137 antibodies. For example, using computationalmodeling techniques, one of skill in the art can screen libraries ofantibodies, antibody fragments, and ligands in silico for moleculescapable of binding specific epitopes on CD137, such as extracellularepitopes of CD137.

Additional techniques can be used to identify antibodies,antigen-binding fragments, and ligands thereof that bind CD137 on thesurface of a cell (e.g., a T cell) and that are internalized by thecell, for instance, by receptor-mediated endocytosis. For example, thein vitro display techniques described above can be adapted to screen forantibodies, antigen-binding fragments thereof, and ligands that bindCD137 on the surface of a hematopoietic stem cell and that aresubsequently internalized. Phage display represents one such techniquethat can be used in conjunction with this screening paradigm. Toidentify anti-CD137 antibodies, fragments thereof, and ligands that bindCD137 and are subsequently internalized by hematopoietic stem cells, oneof skill in the art can use the phage display techniques described inWilliams et al., Leukemia 19:1432-1438, 2005, the disclosure of which isincorporated herein by reference in its entirety. For example, usingmutagenesis methods known in the art, recombinant phage libraries can beproduced that encode antibodies, antibody fragments, such as scFvfragments, Fab fragments, diabodies, triabodies, and ¹⁰Fn3 domains,among others, or ligands that contain randomized amino acid cassettes(e.g., in one or more, or all, of the CDRs or equivalent regions thereofor an antibody or antibody fragment). The framework regions, hinge, Fcdomain, and other regions of the antibodies or antibody fragments may bedesigned such that they are non-immunogenic in humans, for instance, byvirtue of having human germline antibody sequences or sequences thatexhibit only minor variations relative to human germline antibodies.

Using phage display techniques described herein or known in the art,phage libraries containing randomized antibodies, antibody fragments, orligands covalently bound to the phage particles can be incubated withCD137 antigen, for instance, by first incubating the phage library withblocking agents (such as, for instance, milk protein, bovine serumalbumin, and/or IgG so as to remove phage encoding antibodies, fragmentsthereof, or ligands that exhibit non-specific protein binding and phagethat encode antibodies or fragments thereof that bind Fc domains, andthen incubating the phage library with a population of hematopoieticstem cells, which are CD137+. The phage library can be incubated withthe hematopoietic stem cells for a time sufficient to allow CD137specific antibodies, antigen-binding fragments thereof, or ligands tobind cell-surface CD137 and to subsequently be internalized by thehematopoietic stem cells (e.g., from 30 minutes to 6 hours at 4° C.,such as 1 hour at 4° C.). Phage containing antibodies, fragmentsthereof, or ligands that do not exhibit sufficient affinity for CD137 soas to permit binding to, and internalization by, hematopoietic stemcells can subsequently be removed by washing the cells, for instance,with cold (4° C.) 0.1 M glycine buffer at pH 2.8. Phage bound toantibodies, fragments thereof, or ligands that have been internalized bythe hematopoietic stem cells can be identified, for instance, by lysingthe cells and recovering internalized phage from the cell culturemedium. The phage can then be amplified in bacterial cells, for example,by incubating bacterial cells with recovered phage in 2×YT medium usingmethods known in the art. Phage recovered from this medium can then becharacterized, for instance, by determining the nucleic acid sequence ofthe gene(s) encoding the antibodies, fragments thereof, or ligandsinserted within the phage genome. The encoded antibodies, fragmentsthereof, or ligands can subsequently be prepared de novo by chemicalsynthesis (for instance, of antibody fragments, such as scFv fragments,or CD137 ligands) or by recombinant expression (for instance, offull-length antibodies).

The internalizing capacity of the prepared antibodies, fragmentsthereof, or ligands can be assessed, for instance, using radionuclideinternalization assays known in the art. For example, anti-CD137antibodies, fragments thereof, or ligands identified using in vitrodisplay techniques described herein or known in the art can befunctionalized by incorporation of a radioactive isotope, such as ¹⁸F,⁷⁵Br, ⁷⁷Br, ¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, ²¹¹At, ⁶⁷Ga, ¹¹¹In,⁹⁹Tc, ¹⁶⁹Yb, ¹⁸⁶Re, ⁶⁴CU, ⁶⁷′Cu, ¹⁷⁷Lu, ⁷⁷As, ⁷²As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,²¹²Bi, ²¹³Bi, or ²²⁵Ac. For instance, radioactive halogens, such as ¹⁸F,⁷⁵Br, ⁷⁷Br, ¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, ²¹¹At, can beincorporated into antibodies, fragments thereof, or ligands using beads,such as polystyrene beads, containing electrophilic halogen reagents(e.g., Iodination Beads, Thermo Fisher Scientific, Inc., Cambridge,Mass.). Radiolabeled antibodies, fragments thereof, ADCs, or ligands canbe incubated with hematopoietic stem cells for a time sufficient topermit internalization (e.g., from 30 minutes to 6 hours at 4° C., suchas 1 hour at 4° C.). The cells can then be washed to removenon-internalized antibodies or fragments thereof, (e.g., using cold (4°C.) 0.1 M glycine buffer at pH 2.8). Internalized antibodies, fragmentsthereof, or ligands can be identified by detecting the emitted radiation(e.g., γ-radiation) of the resulting hematopoietic stem cells incomparison with the emitted radiation (e.g., γ-radiation) of therecovered wash buffer. The foregoing internalization assays can also beused to characterize ADCs.

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-CD137 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-CLL-1 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-CD137 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.,2003), pp. 245-254, describing expression of antibody fragments in E.coli.) After expression, the antibody may be isolated from the bacterialcell paste in a soluble fraction and can be further purified.

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Drug-Antibody Conjugates Cytotoxins

Antibodies, antigen-binding fragments thereof, and ligands describedherein (e.g., antibodies, antigen-binding fragments thereof, and solubleligands that recognize and bind CD137) can be conjugated (or linked) toa cytotoxins, such as a microtubule-binding agent (for instance,maytansine or a maytansinoid), an amatoxin, pseudomonas exotoxin A,deBouganin, diphtheria toxin, such as α-amanitin, saporin, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof, or another cytotoxic compound described herein or knownin the art in order to promote the depletion of hematopoietic cells,such as a host-reactive T cell, upon administration to a patient. Insome embodiments, the cytotoxic molecule is conjugated to aninternalizing anti-CD137 antibody, antigen-binding fragment thereof orsoluble ligand, such that following the cellular uptake of the antibody,fragment thereof, or soluble ligand, the cytotoxin may access itsintracellular target and mediate hematopoietic cell death. Additionalcytotoxins suitable for use with the compositions and methods describedherein include DNA-intercalating agents, (e.g., anthracyclines), agentscapable of disrupting the mitotic spindle apparatus (e.g., vincaalkaloids, maytansine, maytansinoids, and derivatives thereof), RNApolymerase inhibitors (e.g., an amatoxin, such as α-amanitin, andderivatives thereof), agents capable of disrupting protein biosynthesis(e.g., agents that exhibit rRNA N-glycosidase activity, such as saporinand ricin A-chain), among others known in the art.

Maytansinoids

Anti-CD137 antibodies can be conjugated to a cytotoxin that is amicrotubule binding agent. In some embodiments, the cytotoxin is amaytansine, maytansinoid or maytansinoid analog. Maytansinoids aremicrotubule binding agents that prohibit tubulin polymerization.Examples of suitable maytansinoids include esters of maytansinol,synthetic maytansinol, and maytansinol analogs and derivatives. Includedare any drugs that inhibit microtubule formation and that are highlytoxic to mammalian cells, as are maytansinoids, maytansinol, andmaytansinol analogs, and derivatives.

Examples of suitable maytansinol esters include those having a modifiedaromatic ring and those having modifications at other positions. Suchsuitable maytansinoids are disclosed in U.S. Pat. Nos. 4,137,230;4,151,042; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,362,663; 4,364,866;4,424,219; 4,450,254; 4,322,348; 4,362,663; 4,371,533; 5,208,020;5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497; and7,473,796, the disclosures of each of which are incorporated herein byreference as they pertain to maytansinoids and derivatives thereof.

In some embodiments, the immunoconjugates (ADCs) of the inventionutilize the thiol-containing maytansinoid (DM1), formally termedN²′-deacetyl-N²′-(3-mercapto-1-oxopropyl)-maytansine, as the cytotoxicagent. DM1 is represented by the following structural formula:

In another embodiment, the conjugates of the present invention utilizethe thiol-containing maytansinoidN²′-deacetyl-N²′(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4)as the cytotoxic agent. DM4 is represented by the following structuralformula:

Another maytansinoid comprising a side chain that contains a stericallyhindered thiol bond isN²′-deacetyl-N-2′(4-mercapto-1-oxopentyl)-maytansine (termed DM3),represented by the following structural formula (III):

Each of the maytansinoids taught in U.S. Pat. Nos. 5,208,020 and7,276,497, can also be used in the conjugate of the present invention.In this regard, the entire disclosure of U.S. Pat. Nos. 5,208,020 and7,276,697 is incorporated herein by reference.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. In some embodiments, the C-3 position servesas the position to chemically link the linking moiety, and in someparticular embodiments, the C-3 position of maytansinol serves as theposition to chemically link the linking moiety.

The invention also includes various isomers and mixtures ofmaytansinoids and conjugates. Certain compounds and conjugates of thepresent invention may exist in various stereoisomeric, enantiomeric, anddiastereomeric forms. Several descriptions for producing suchantibody-maytansinoid conjugates are provided in U.S. Pat. Nos.5,208,020, 5,416,064 6,333,410, 6,441,163, 6,716,821, and 7,368,565,each of which is incorporated herein in its entirety.

A therapeutically effective number of maytansinoid molecules bound perantibody molecule can be determined by measuring spectrophotometricallythe ratio of the absorbance at 252 nm and 280 nm. An average of 3 to 4maytansinoid molecules conjugated per antibody molecule can enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody, although one molecule of toxin/antibodycan enhance cytotoxicity over antibody alone. The average number ofmaytansinoid molecules/antibody or antigen binding fragment thereof, orsoluble ligand, can be, for example, 1-10 or 2-5.

Amatoxins

In some embodiments, the cytotoxin is an amatoxin or derivative thereof,such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin,amaninamide, amanullin, amanullinic acid, or proamanullin. For instance,suitable cytotoxins that may be conjugated to an antibody,antigen-binding fragment thereof, or soluble ligand described hereininclude an amatoxin or derivative thereof represented by formula (IV)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is L-Z;

R_(D) is optionally substituted C₁-C₆ alkyl, optionally substitutedC₁-C₆ heteroalkyl, optionally substituted C₂-C₆ alkenyl, optionallysubstituted C₂-C₆ heteroalkenyl, optionally substituted C₂-C₆ alkynyl,optionally substituted C₂-C₆ heteroalkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, or optionally substituted heteroaryl;

L is a linker, such as optionally substituted C₁-C₆ alkylene, optionallysubstituted C₁-C₆ heteroalkylene, optionally substituted C₂-C₆alkenylene, optionally substituted C₂-C₆ heteroalkenylene, optionallysubstituted C₂-C₆ alkynylene, optionally substituted C₂-C₆heteroalkynylene, optionally substituted cycloalkylene, optionallysubstituted heterocycloalkylene, optionally substituted arylene, oroptionally substituted heteroarylene; and

Z is a chemical moiety that forms a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within anantibody, antigen-binding fragment thereof, or soluble ligand that bindsCD137.

In some embodiments, the cytotoxin contains one R_(C) substituent.

In some embodiments, the cytotoxin is an amatoxin or derivative thereofrepresented by formula (IVA)

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup;

R₃ is H, R_(C), or R_(D);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D);

R₉ is H, OH, OR_(C), or OR_(D);

X is —S—, —S(O)—, or —SO₂—;

R_(C) is L-Z;

R_(D) is optionally substituted C₁-C₆ alkyl, optionally substitutedC₁-C₆ heteroalkyl, optionally substituted C₂-C₆ alkenyl, optionallysubstituted C₂-C₆ heteroalkenyl, optionally substituted C₂-C₆ alkynyl,optionally substituted C₂-C₆ heteroalkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, or optionally substituted heteroaryl;

L is optionally substituted C₁-C₆ alkylene, optionally substituted C₁-C₆heteroalkylene, optionally substituted C₂-C₆ alkenylene, optionallysubstituted C₂-C₆ heteroalkenylene, optionally substituted C₂-C₆alkynylene, optionally substituted C₂-C₆ heteroalkynylene, optionallysubstituted cycloalkylene, optionally substituted heterocycloalkylene,optionally substituted arylene, or optionally substituted heteroarylene;

Z is a chemical moiety that forms a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within anantibody, antigen-binding fragment thereof, or soluble ligands thatbinds CD137; and

wherein the cytotoxin contains one R_(C) substituent.

In some embodiments, R_(A) and R_(B), together with the oxygen atoms towhich they are bound, combine to form:

wherein Y is selected from O, S, NR_(E), and CR_(E)R_(E′), and

R_(E) and R_(E′) are each independently optionally substituted C₁-C₆alkylene-R_(C), optionally substituted C₁-C₆ heteroalkylene-R_(C),optionally substituted C₂-C₆ alkenylene-R_(C), optionally substitutedC₂-C₆ heteroalkenylene-R_(C), optionally substituted C₂-C₆alkynylene-R_(C), optionally substituted C₂-C₆ heteroalkynylene-R_(C),optionally substituted cycloalkylene-R_(C), optionally substitutedheterocycloalkylene-R_(C), optionally substituted arylene-R_(C), oroptionally substituted heteroarylene-R_(C).

In some embodiments, the cytotoxin is an amatoxin or derivative thereofrepresented by formula (IA), wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃ is H or R_(C);

R₄ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₅ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₆ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₇ is H, OH, OR_(C), OR_(D), R_(C), or R_(D);

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) and R_(D) are each as defined above. Toxins useful inconjunction with the conjugates described herein include those thatcontain an amatoxin or derivative thereof represented by formula (IVA),

wherein R₁ is H, OH, OR_(A), or OR_(C);

R₂ is H, OH, OR_(B), or OR_(C);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃ is H or R_(C);

R₄ and R₅ are each independently H, OH, OR_(C), R_(C), or OR_(D);

R₆ and R₇ are each H;

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) is as defined above.

Toxins useful in conjunction with the conjugates described hereininclude those that contain an amatoxin or derivative thereof representedby formula (IVA),

wherein R₁ is H, OH, or OR_(A);

R₂ is H, OH, or OR_(B);

R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form:

R₃, R₄, R₆, and R₇ are each H;

R₅ is OR_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in US Patent Application Publication No.2016/0002298, the disclosure of which is incorporated herein byreference in its entirety.

Toxins useful in conjunction with the conjugates described hereininclude those that contain an amatoxin or derivative thereof representedby formula (IVA),

wherein R₁ and R₂ are each independently H or OH;

R₃ is R_(C);

R₄, R₆, and R₇ are each H;

R₅ is H, OH, or OC₁-C₆ alkyl;

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in US Patent Application Publication No.2014/0294865, the disclosure of which is incorporated herein byreference in its entirety.

Toxins useful in conjunction with the conjugates described hereininclude those that contain an amatoxin or derivative thereof representedby formula (IA),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H, OH, OR_(C), or R_(C);

R₈ is OH or NH₂;

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in US Patent Application Publication No.2015/0218220, the disclosure of which is incorporated herein byreference in its entirety.

Toxins useful in conjunction with the conjugates described hereininclude those that contain an amatoxin or derivative thereof representedby formula (IVA),

wherein R₁ and R₂ are each independently H or OH;

R₃, R₆, and R₇ are each H;

R₄ and R₅ are each independently H or OH;

R₈ is OH, NH₂, OR_(C), or NHR_(C);

R₉ is H or OH; and

wherein R_(C) is as defined above. Such amatoxin conjugates aredescribed, for example, in U.S. Pat. Nos. 9,233,173 and 9,399,681, thedisclosures of each of which are incorporated herein by reference intheir entirety.

In some embodiments, antibodies or antigen-binding fragments thereof,described herein are conjugated to an amatoxin, such as α-amanitin, or avariant thereof. For instance, in some embodiments, antibodies orantigen-binding fragments, described herein (e.g., antibodies orantigen-binding fragments that recognize and bind CD137) are conjugatedto an α-amanitin compound represented by formula (II):

wherein X is S, SO, or SO₂; R₁ is H or a linker covalently bound to theantibody, antigen-binding fragment thereof or ligand; and R₂ is H or alinker covalently bound to the antibody, antigen-binding fragmentthereof or ligand; wherein when R₁ is H, R₂ is the linker, and when R₂is H, R₁ is the linker.

In some embodiments, the cytotoxin is an α-amanitin. In someembodiments, the α-amanitin is a compound of formula IV. In someembodiments, the α-amanitin of formula IV is attached to an anti-CD137antibody via a linker L. The linker L may be attached to the α-amanitinof formula IV at any one of several possible positions (e.g., any ofR¹-R⁹). In some embodiments, the linker is attached at position R¹. Insome embodiments, the linker is attached at position R². In someembodiments, the linker is attached at position R³. In some embodiments,the linker is attached at position R⁴. In some embodiments, the linkeris attached at position R⁵. In some embodiments, the linker is attachedat position R⁶. In some embodiments, the linker is attached at positionR⁷. In some embodiments, the linker is attached at position R⁸. In someembodiments, the linker is attached at position R⁹. In some embodiments,the linker includes a hydrazine, a disulfide, a thioether or adipeptide. In some embodiments, the linker includes a dipeptide selectedfrom Val-Ala and Val-Cit. In some embodiments, the linker includes apara-aminobenzyl group (PAB). In some embodiments, the linker includesthe moiety PAB-Cit-Val. In some embodiments, the linker includes themoiety PAB-Ala-Val. In some embodiments, the linker includes a—((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6. In someembodiments, the linker is -PAB-Cit-Val-((C═O)(CH₂)_(n)—. In someembodiments, the linker is -PAB-Ala-Val-((C═O)(CH₂)_(n)—.

In some embodiments, the cytotoxin is a β-amanitin. In some embodiments,the 3-amanitin is a compound of formula IV. In some embodiments, theβ-amanitin of formula IV is attached to an anti-CD137 antibody via alinker L. The linker L may be attached to the β-amanitin of formula IVat any one of several possible positions (e.g., any of R¹-R⁹). In someembodiments, the linker is attached at position R¹. In some embodiments,the linker is attached at position R². In some embodiments, the linkeris attached at position R³. In some embodiments, the linker is attachedat position R⁴. In some embodiments, the linker is attached at positionR⁵. In some embodiments, the linker is attached at position R⁶. In someembodiments, the linker is attached at position R⁷. In some embodiments,the linker is attached at position R⁸. In some embodiments, the linkeris attached at position R⁹. In some embodiments, the linker includes ahydrazine, a disulfide, a thioether or a dipeptide. In some embodiments,the linker includes a dipeptide selected from Val-Ala and Val-Cit. Insome embodiments, the linker includes a para-aminobenzyl group (PAB). Insome embodiments, the linker includes the moiety PAB-Cit-Val. In someembodiments, the linker includes the moiety PAB-Ala-Val. In someembodiments, the linker includes a —((C═O)(CH₂)_(n)— unit, wherein n isan integer from 1-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—.

In some embodiments, the cytotoxin is a γ-amanitin. In some embodiments,the γ-amanitin is a compound of formula IV. In some embodiments, theγ-amanitin of formula IV is attached to an anti-CD137 antibody via alinker L. The linker L may be attached to the γ-amanitin of formula IVat any one of several possible positions (e.g., any of R¹-R⁹). In someembodiments, the linker is attached at position R¹. In some embodiments,the linker is attached at position R². In some embodiments, the linkeris attached at position R³. In some embodiments, the linker is attachedat position R⁴. In some embodiments, the linker is attached at positionR⁵. In some embodiments, the linker is attached at position R⁶. In someembodiments, the linker is attached at position R⁷. In some embodiments,the linker is attached at position R⁸. In some embodiments, the linkeris attached at position R⁹. In some embodiments, the linker includes ahydrazine, a disulfide, a thioether or a dipeptide. In some embodiments,the linker includes a dipeptide selected from Val-Ala and Val-Cit. Insome embodiments, the linker includes a para-aminobenzyl group (PAB). Insome embodiments, the linker includes the moiety PAB-Cit-Val. In someembodiments, the linker includes the moiety PAB-Ala-Val. In someembodiments, the linker includes a —((C═O)(CH₂)_(n)— unit, wherein n isan integer from 1-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—.

In some embodiments, the cytotoxin is a ε-amanitin. In some embodiments,the ε-amanitin is a compound of formula IV. In some embodiments, theε-amanitin of formula IV is attached to an anti-CD137 antibody via alinker L. The linker L may be attached to the ε-amanitin of formula IVat any one of several possible positions (e.g., any of R¹-R⁹). In someembodiments, the linker is attached at position R¹. In some embodiments,the linker is attached at position R². In some embodiments, the linkeris attached at position R³. In some embodiments, the linker is attachedat position R⁴. In some embodiments, the linker is attached at positionR⁵. In some embodiments, the linker is attached at position R⁶. In someembodiments, the linker is attached at position R⁷. In some embodiments,the linker is attached at position R⁸. In some embodiments, the linkeris attached at position R⁹. In some embodiments, the linker includes ahydrazine, a disulfide, a thioether or a dipeptide. In some embodiments,the linker includes a dipeptide selected from Val-Ala and Val-Cit. Insome embodiments, the linker includes a para-aminobenzyl group (PAB). Insome embodiments, the linker includes the moiety PAB-Cit-Val. In someembodiments, the linker includes the moiety PAB-Ala-Val. In someembodiments, the linker includes a —((C═O)(CH₂)_(n)— unit, wherein n isan integer from 1-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—.

In some embodiments, the cytotoxin is an amanin. In some embodiments,the amanin is a compound of formula IV. In some embodiments, the amaninof formula IV is attached to an anti-CD137 antibody via a linker L. Thelinker L may be attached to the amanin of formula IV at any one ofseveral possible positions (e.g., any of R¹-R⁹). In some embodiments,the linker is attached at position R¹. In some embodiments, the linkeris attached at position R². In some embodiments, the linker is attachedat position R³. In some embodiments, the linker is attached at positionR⁴. In some embodiments, the linker is attached at position R⁵. In someembodiments, the linker is attached at position R⁶. In some embodiments,the linker is attached at position R⁷. In some embodiments, the linkeris attached at position R⁸. In some embodiments, the linker is attachedat position R⁹. In some embodiments, the linker includes a hydrazine, adisulfide, a thioether or a dipeptide. In some embodiments, the linkerincludes a dipeptide selected from Val-Ala and Val-Cit. In someembodiments, the linker includes a para-aminobenzyl group (PAB). In someembodiments, the linker includes the moiety PAB-Cit-Val. In someembodiments, the linker includes the moiety PAB-Ala-Val. In someembodiments, the linker includes a —((C═O)(CH₂)_(n)-unit, wherein n isan integer from 1-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—.

In some embodiments, the cytotoxin is an amaninamide. In someembodiments, the amaninamide is a compound of formula IV. In someembodiments, the amaninamide of formula IV is attached to an anti-CD137antibody via a linker L. The linker L may be attached to the amaninamideof formula IV at any one of several possible positions (e.g., any ofR¹-R⁹). In some embodiments, the linker is attached at position R¹. Insome embodiments, the linker is attached at position R². In someembodiments, the linker is attached at position R³. In some embodiments,the linker is attached at position R⁴. In some embodiments, the linkeris attached at position R⁵. In some embodiments, the linker is attachedat position R⁶. In some embodiments, the linker is attached at positionR⁷. In some embodiments, the linker is attached at position R⁸. In someembodiments, the linker is attached at position R⁹. In some embodiments,the linker includes a hydrazine, a disulfide, a thioether or adipeptide. In some embodiments, the linker includes a dipeptide selectedfrom Val-Ala and Val-Cit. In some embodiments, the linker includes apara-aminobenzyl group (PAB). In some embodiments, the linker includesthe moiety PAB-Cit-Val. In some embodiments, the linker includes themoiety PAB-Ala-Val. In some embodiments, the linker includes a—((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6. In someembodiments, the linker is -PAB-Cit-Val-((C═O)(CH₂)_(n)—. In someembodiments, the linker is -PAB-Ala-Val-((C═O)(CH₂)_(n)—.

In some embodiments, the cytotoxin is an amanullin. In some embodiments,the amanullin is a compound of formula IV. In some embodiments, theamanullin of formula IV is attached to an anti-CD137 antibody via alinker L. The linker L may be attached to the amanullin of formula IV atany one of several possible positions (e.g., any of R¹-R⁹). In someembodiments, the linker is attached at position R¹. In some embodiments,the linker is attached at position R². In some embodiments, the linkeris attached at position R³. In some embodiments, the linker is attachedat position R⁴. In some embodiments, the linker is attached at positionR⁵. In some embodiments, the linker is attached at position R⁶. In someembodiments, the linker is attached at position R⁷. In some embodiments,the linker is attached at position R⁸. In some embodiments, the linkeris attached at position R⁹. In some embodiments, the linker includes ahydrazine, a disulfide, a thioether or a dipeptide. In some embodiments,the linker includes a dipeptide selected from Val-Ala and Val-Cit. Insome embodiments, the linker includes a para-aminobenzyl group (PAB). Insome embodiments, the linker includes the moiety PAB-Cit-Val. In someembodiments, the linker includes the moiety PAB-Ala-Val. In someembodiments, the linker includes a —((C═O)(CH₂)_(n)— unit, wherein n isan integer from 1-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—.

In some embodiments, the cytotoxin is an amanullinic acid. In someembodiments, the amanullinic acid is a compound of formula IV. In someembodiments, the amanullinic acid of formula IV is attached to ananti-CD137 antibody via a linker L. The linker L may be attached to theamanullinic acid of formula IV at any one of several possible positions(e.g., any of R¹-R⁹). In some embodiments, the linker is attached atposition R¹. In some embodiments, the linker is attached at position R².In some embodiments, the linker is attached at position R³. In someembodiments, the linker is attached at position R⁴. In some embodiments,the linker is attached at position R⁵. In some embodiments, the linkeris attached at position R⁶. In some embodiments, the linker is attachedat position R⁷. In some embodiments, the linker is attached at positionR⁸. In some embodiments, the linker is attached at position R⁹. In someembodiments, the linker includes a hydrazine, a disulfide, a thioetheror a dipeptide. In some embodiments, the linker includes a dipeptideselected from Val-Ala and Val-Cit. In some embodiments, the linkerincludes a para-aminobenzyl group (PAB). In some embodiments, the linkerincludes the moiety PAB-Cit-Val. In some embodiments, the linkerincludes the moiety PAB-Ala-Val. In some embodiments, the linkerincludes a —((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6. Insome embodiments, the linker is -PAB-Cit-Val-((C═O)(CH₂)_(n)—. In someembodiments, the linker is -PAB-Ala-Val-((C═O)(CH₂)_(n)—.

In some embodiments, the cytotoxin is a proamanullin. In someembodiments, the proamanullin is a compound of formula IV. In someembodiments, the proamanullin of formula IV is attached to an anti-CD137antibody via a linker L. The linker L may be attached to theproamanullin of formula IV at any one of several possible positions(e.g., any of R¹-R⁹). In some embodiments, the linker is attached atposition R¹. In some embodiments, the linker is attached at position R².In some embodiments, the linker is attached at position R³. In someembodiments, the linker is attached at position R⁴. In some embodiments,the linker is attached at position R⁵. In some embodiments, the linkeris attached at position R⁶. In some embodiments, the linker is attachedat position R⁷. In some embodiments, the linker is attached at positionR⁸. In some embodiments, the linker is attached at position R⁹. In someembodiments, the linker includes a hydrazine, a disulfide, a thioetheror a dipeptide. In some embodiments, the linker includes a dipeptideselected from Val-Ala and Val-Cit. In some embodiments, the linkerincludes a para-aminobenzyl group (PAB). In some embodiments, the linkerincludes the moiety PAB-Cit-Val. In some embodiments, the linkerincludes the moiety PAB-Ala-Val. In some embodiments, the linkerincludes a —((C═O)(CH₂)_(n)— unit, wherein n is an integer from 1-6. Insome embodiments, the linker is -PAB-Cit-Val-((C═O)(CH₂)_(n)—. In someembodiments, the linker is -PAB-Ala-Val-((C═O)(CH₂)_(n)—.

Antibodies, antigen-binding fragments, and ligands for use with thecompositions and methods described herein can be conjugated to anamatoxin, such as α-amanitin or a variant thereof using conjugationtechniques known in the art or described herein. For instance,antibodies, antigen-binding fragments thereof, and ligands thatrecognize and bind CD137 can be conjugated to α-amanitin or a variantthereof, as described in US 2015/0218220, the disclosure of which isincorporated herein by reference as it pertains, for example, toamatoxins, such as α-amanitin and variants thereof, as well as covalentlinkers that can be used for covalent conjugation.

Exemplary antibody-drug conjugates and ligand-drug conjugates useful inconjunction with the methods described herein may be formed by thereaction of an antibody, antigen-binding fragment thereof, or ligandwith an amatoxin that is conjugated to a linker containing a substituentsuitable for reaction with a reactive residue on the antibody,antigen-binding fragment thereof, or ligand. Amatoxins that areconjugated to a linker containing a substituent suitable for reactionwith a reactive residue on the antibody, antigen-binding fragmentthereof, or ligand include, without limitation,7′C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(6-(maleimido)hexanamido)piperidin-1-yl)-amatoxin;7′C-(4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-amatoxin;7′C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(3-carboxypropanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amatoxin;7′C-(4-(3-(maleimido)propanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(4-(maleimido)butanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(3-((6-(6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)-amatoxin;7′-3(6(4(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7′C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)-amatoxin;7′C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)-amatoxin;7′C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-1-yl)-amatoxin;7′C-((4-(6-(maleimido)hexanamido)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;(R)-7′C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;(S)-7′C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-(4-(6(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(3-carboxypropanamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-(4-(6(maleimido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-(4 (2-(maleimido)acetyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(3-(maleimido)propanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((2-(6-(4((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amatoxin;7′C-((3-(2-(6-(maleimido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxin;7′C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amatoxin;7′C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1yl)methyl)-amatoxin;7′C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxin;7′C-(((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxin;7′C-(((4-(6-(maleimido)-N-methylhexanamido)butyl(methyl)amino)methyl)-amatoxin;7′C-((2-(2-(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxin;7′C-((2-(2-(6(4((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxin;7′C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(3(2-(aminooxy)acetamido)propanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-(4-(2 (aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amatoxin;7′C-((2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((2-(4(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidin-1-yl)methyl)-amatoxin;7′C-(((2-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxin; 7′C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)butyl)(methyl) amino)methyl)-amatoxin; 7′C-(3((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amatoxin;7′C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-amatoxin;7′C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7′C-((4-(2-(3-(pyridine-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;6′O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxin;6′O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-amatoxin;6′O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amatoxin;6′O-((6-(maleimido)hexyl)carbamoyl)-amatoxin;6′O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amatoxin;6′O-(6-(2-bromoacetamido)hexyl)-amatoxin;7′C-(4-(6-(azido)hexanamido)piperidin-1-yl)-amatoxin;7′C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amatoxin;7′C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;7′C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;6′O-(6-(6-(11,12-didehydro-5,6-dihydro-dibenz[b,f]azocin-5-yl)-6-oxohexanamido)hexyl)-amatoxin;6′O-(6-(hex-5-ynoylamino)hexyl)-amatoxin;6′O-(6-(2-(aminooxy)acetylamido)hexyl)-amatoxin;6′O-((6-aminooxy)hexyl)-amatoxin; and6′O-(6-(2-iodoacetamido)hexyl)-amatoxin. The foregoing linkers, amongothers useful in conjunction with the compositions and methods describedherein, are described, for example, in US Patent Application PublicationNo. 2015/0218220, the disclosure of which is incorporated herein byreference in its entirety.

Additional cytotoxins that can be conjugated to antibodies,antigen-binding fragments thereof, and ligands that recognize and bindCD137 for use in treatment of GVHD or an autoimmune disease include,without limitation, 5-ethynyluracil, abiraterone, acylfulvene,adecypenol, adozelesin, aldesleukin, altretamine, ambamustine, amidox,amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide,anastrozole, andrographolide, angiogenesis inhibitors, antarelix,anti-dorsalizing morphogenetic protein-1, antiandrogen, prostaticcarcinoma, antiestrogen, antineoplaston, antisense oligonucleotides,aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators,apurinic acid, asulacrine, atamestane, atrimustine, axinastatin 1,axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatinIII derivatives, balanol, batimastat, BCR/ABL antagonists,benzochlorins, benzoylstaurosporine, beta lactam derivatives,beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitors,bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistrateneA, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine,budotitane, buthionine sulfoximine, calcipotriol, calphostin C,camptothecin derivatives (e.g., 10-hydroxy-camptothecin), capecitabine,carboxamide-amino-triazole, carboxyamidotriazole, carzelesin, caseinkinase inhibitors, castanospermine, cecropin B, cetrorelix, chlorins,chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine,clomifene and analogues thereof, clotrimazole, collismycin A,collismycin B, combretastatin A4, combretastatin analogues, conagenin,crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives,curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabineocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine,dehydrodidemnin B, 2′deoxycoformycin (DCF), deslorelin, dexifosfamide,dexrazoxane, dexverapamil, diaziquone, didemnin B, didox,diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin,diphenyl spiromustine, discodermolide, docosanol, dolasetron,doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen,ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur,epothilones, epithilones, epristeride, estramustine and analoguesthereof, etoposide, etoposide 4′-phosphate (also referred to asetopofos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim,finasteride, flavopiridol, flezelastine, fluasterone, fludarabine,fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin,fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine,ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors,hepsulfam, homoharringtonine (HHT), hypericin, ibandronic acid,idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones,imiquimod, immunostimulant peptides, iobenguane, iododoxorubicin,ipomeanol, irinotecan, iroplact, irsogladine, isobengazole,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol,lonidamine, losoxantrone, loxoribine, lurtotecan, lutetium texaphyrin,lysofylline, masoprocol, maspin, matrix metalloproteinase inhibitors,menogaril, rnerbarone, meterelin, methioninase, metoclopramide, MIFinhibitor, ifepristone, miltefosine, mirimostim, mithracin, mitoguazone,mitolactol, mitomycin and analogues thereof, mitonafide, mitoxantrone,mofarotene, molgramostim, mycaperoxide B, myriaporone, N-acetyldinaline,N-substituted benzamides, nafarelin, nagrestip, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide,nisamycin, nitrullyn, octreotide, okicenone, onapristone, ondansetron,oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and analoguesthereof, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol,panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosanpolysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide,phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin,porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed,rhizoxin, rogletimide, rohitukine, rubiginone B1, ruboxyl, safingol,saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparfosicacid, spicamycin D, spiromustine, stipiamide, sulfinosine, tallimustine,tegafur, temozolomide, teniposide, thaliblastine, thiocoraline,tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine,vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb, amongothers.

Linkers for Chemical Conjugation

A variety of linkers can be used to conjugate antibodies,antigen-binding fragments, and ligands described herein (e.g.,antibodies, antigen-binding fragments thereof, and soluble ligands thatrecognize and bind CD137) with a cytotoxic molecule. Suitable linkersinclude those that may be cleaved, for instance, by enzymatichydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysisunder basic conditions, oxidation, disulfide reduction, nucleophiliccleavage, or organometallic cleavage (see, for example, Leriche et al.,Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which isincorporated herein by reference as it pertains to linkers suitable forcovalent conjugation). Examples of linkers useful for the synthesis ofdrug-antibody and drug-ligand conjugates include those that containelectrophiles, such as Michael acceptors (e.g., maleimides), activatedesters, electron-deficient carbonyl compounds, and aldehydes, amongothers, suitable for reaction with nucleophilic substituents presentwithin antibodies, antigen-binding fragments, and ligands such as amineand thiol moieties. For instance, linkers suitable for the synthesis ofdrug-antibody and drug-ligand conjugates include, without limitation,succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC),N-succinimidyl iodoacetate (SIA), sulfo-SMCC,m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, andsuccinimidyl iodoacetate, among others described, for instance, Liu etal., 18:690-697, 1979, the disclosure of which is incorporated herein byreference as it pertains to linkers for chemical conjugation. Additionallinkers include the non-cleavable maleimidocaproyl linkers, which areparticularly useful for the conjugation of microtubule-disrupting agentssuch as auristatins, are described by Doronina et al., BioconjugateChem. 17:14-24, 2006, the disclosure of which is incorporated herein byreference as it pertains to linkers for chemical conjugation. Additionallinkers suitable for the synthesis of drug-antibody and drug-ligandconjugates as described herein include those capable of releasing acytotoxin by a 1,6-elimination process, such as p-aminobenzyl alcohol(PABC), 6-maleimidohexanoic acid, pH-sensitive carbonates, and otherreagents described in Jain et al., Pharm. Res. 32:3526-3540, 2015, thedisclosure of which is incorporated herein by reference in its entirety.

Linkers that can be used to conjugate an antibody, antigen-bindingfragment thereof, or ligand to a cytotoxic agent include those that arecovalently bound to the cytotoxic agent on one end of the linker and, onthe other end of the linker, contain a chemical moiety formed from acoupling reaction between a reactive substituent present on the linkerand a reactive substituent present within the antibody, antigen-bindingfragment thereof, or ligand that binds CD137. Reactive substituents thatmay be present within an antibody, antigen-binding fragment thereof, orligand that binds CD137 include, without limitation, hydroxyl moietiesof serine, threonine, and tyrosine residues; amino moieties of lysineresidues; carboxyl moieties of aspartic acid and glutamic acid residues;and thiol moieties of cysteine residues, as well as propargyl, azido,haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl),haloalkyl, and haloheteroalkyl moieties of non-naturally occurring aminoacids. Linkers useful in conjunction with the antibody-drug andligand-conjugates described herein include, without limitation, linkerscontaining chemical moieties formed by coupling reactions as depicted inTable 1, below. Curved lines designate points of attachment to theantibody, antigen-binding fragment, or ligand and the cytotoxicmolecule, respectively.

TABLE 1 Exemplary chemical moieties formed by coupling reactions in theformation of antibody- drug conjugates Exemplary Coupling ReactionsChemical Moiety Formed by Coupling Reactions [3 + 2] Cycloaddition

[3 + 2] Cycloaddition

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Esterification

[3 + 2] Cycloaddition, Etherification

[3 + 2] Cycloaddition

Michael addition

Michael addition

Imine condensation, Amidation

Imine condensation

Disulfide formation

Thiol alkylation

Condensation, Michael addition

In some embodiments, the ADC comprises an anti-CD137 antibody conjugatedto a toxin via linker, wherein the linker includes a hydrazine, adisulfide, a thioether or a dipeptide. In some embodiments, the linkerincludes a dipeptide selected from Val-Ala and Val-Cit. In someembodiments, the linker includes a para-aminobenzyl group (PAB). In someembodiments, the linker includes the moiety PAB-Cit-Val. In someembodiments, the linker includes the moiety PAB-Ala-Val. In someembodiments, the linker includes a —((C═O)(CH₂)_(n)— unit, wherein n isan integer from 1-6. In some embodiments, the linker is-PAB-Cit-Val-((C═O)(CH₂)_(n)—. In some embodiments, the linker is-PAB-Ala-Val-((C═O)(CH₂)_(n)—.

Antibody Pharmacokinetic Profile

In some embodiments, the antibody, antigen-binding fragment thereof, ordrug-antibody conjugate has a defined serum half-life. Antibodies,antigen-binding fragments thereof, and conjugates useful in the methodsherein include those that have a serum half-life, for example, from 1-24hours. In some embodiments, the transplant is administered prior, at thesame time or after the antibody, antigen-binding fragment thereof,drug-antibody conjugate, when the level of the circulating antibody isat a therapeutically effective level. Pharmacokinetic analysis bymeasurement of serum levels can be performed by assays known in the art.

Routes of Administration and Dosing

Antibodies, antigen-binding fragments thereof, ADCs, and ligandsdescribed herein can be administered to a patient (e.g., a human patientsuffering from or at risk for GVHD or an autoimmune disease) in avariety of dosage forms. For instance, antibodies, antigen-bindingfragments thereof, ADCs, and ligands described herein can beadministered to a patient suffering from or at risk for GVHD in the formof an aqueous solution, such as an aqueous solution containing one ormore pharmaceutically acceptable excipients. Suitable pharmaceuticallyacceptable excipients for use with the compositions and methodsdescribed herein include viscosity-modifying agents. The aqueoussolution may be sterilized using techniques known in the art.

Pharmaceutical formulations comprising anti-CD137 ADCs as describedherein are prepared by mixing such ADC with one or more optionalpharmaceutically acceptable carriers (Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Pharmaceutically acceptable carriersare generally nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG).

The antibodies, antigen-binding fragments, ADCs, and ligands describedherein may be administered by a variety of routes, such as orally,transdermally, subcutaneously, intranasally, intravenously,intramuscularly, intraocularly, or parenterally. The most suitable routefor administration in any given case will depend on the particularantibody, antigen-binding fragment administered, or ADC, the patient,pharmaceutical formulation methods, administration methods (e.g.,administration time and administration route), the patient's age, bodyweight, sex, severity of the diseases being treated, the patient's diet,and the patient's excretion rate.

The effective dose of an antibody, antigen-binding fragment thereof,ADC, or ligand described herein can range, for example from about 0.001to about 100 mg/kg of body weight per single (e.g., bolus)administration, multiple administrations, or continuous administration,or to achieve an optimal serum concentration (e.g., a serumconcentration of 0.0001-5000 μg/mL) of the antibody, antigen-bindingfragment thereof, ADC, or soluble ligand. The dose may be administeredone or more times (e.g., 2-10 times) per day, week, or month to asubject (e.g., a human) suffering from or at risk for GVHD or anautoimmune disease. The antibody, antigen-binding fragment thereof, ADC,or ligand can be administered in an amount sufficient to reduce thequantity of host-reactive T cells, for example, by 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or more prior to hematopoietic stem celltransplant.

Methods of Treatment

The compositions and methods described herein may be used to depleteactivated T cells that are associated with graft failure and autoimmunediseases in order to achieve transplant tolerance. The compositions andmethods described herein are particularly useful for preventing andtreating GVHD and autoimmune diseases. The methods and compositionsdisclosed herein are also useful in reducing the risk of transplantfailure in a human patient receiving an allogenic transplant. Thepreferred subject is human. The amount of antibody, antibody-drugconjugate, or ligand-drug conjugate administered should be sufficient todeplete cells, e.g., activated T cells, that promote GVHD or autoimmunedisease. The determination of a therapeutically effective dose is withinthe capability of practitioners in this art, however, as an example, inembodiments of the method described herein utilizing systemicadministration of an antibody for the treatment of GHVD or autoimmunedisease, an effective human dose will be in the range of 0.1-150 mg/kg(e.g., 5 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 150mg/kg etc.). The route of administration may affect the recommendeddose. Repeated systemic doses are contemplated in order to maintain aneffective level, e.g., to attenuate or inhibit GVHD or autoimmunedisease, depending on the mode of administration adopted.

The antibody, antibody-drug conjugate, or ligand-drug conjugate can beadministered to the human patient in need prior to, concomitantly with,or after transplantation of cells or a solid organ to the patient. Inone embodiment, an anti-CD137 ADC is administered to the human patientin need thereof prior to (e.g., 3 days before, 2 days before, 12 hoursbefore) transplantation of cells or a solid organ. In one embodiment, ananti-CD137 ADC is administered to the human patient in need thereofafter (e.g., 1 days after, 2 days after, 3 days after, or 4 days after)transplantation of cells or a solid organ. A single dose of ananti-CD137 ADC may be administered to the human patient either prior to,concomitantly with, or after transplantation of cells or an organ, wheresuch single dose is sufficient to treat or prevent GVHD or graftfailure.

Anti-CD137 ADCs may be used as an alternative to traditional agents(e.g., chemotherapy and/or radiation) used to promote acceptance of atransplant, including an allogeneic transplant. Traditional agentsgenerally reduce a patient's immune response in order to promoteengraftment and acceptance of the transplanted cells or organ. Themethods and compositions described herein provide a more selectivetherapy that allows much of the patient's immune system to remainintact, while targeting and depleting CD137 expressing activated Tcells. Thus, the ability of anti-CD137 ADCs disclosed herein toselectively deplete activated T cells provides an advantageous therapyover traditional therapy in the context of transplantation given that,in particular, allo-activated immune cells can be targeted and depletedin order to achieve successful transplantation of cells or a solidorgan.

The methods and compositions disclosed herein may be used to prevent ortreat graft failure. Graft failure or graft rejection, including failureafter allogeneic hematopoietic stem cell transplantation, may bemanifested generally as either lack of initial engraftment of donorcells, or loss of donor cells after initial engraftment (for review seeMattsson et al. (2008) Biol Blood Marrow Transplant. 14(Suppl 1):165-170). Compositions and methods disclosed herein may be used todeplete CD137 expressing activated T cells in a graft or transplantationscenario where graft failure is of concern, e.g., where the humanpatient is at risk of developing graft failure following transplantationof a solid organ or cells, particularly where the transplanted cells ororgan is allogeneic.

In one embodiment, the anti-CD137 antibody, antibody-drug conjugate, orligand-drug conjugate is used to deplete CD137 expressing donor cells,e.g., activated T cells expressing CD137, by contacting the cells, graftor solid organ with the anti-CD137 antibody, antibody-drug conjugate, orligand-drug conjugate prior to transplantation of the cells, graft ororgan to a human patient. In one embodiment, the cells, graft or organare allogeneic.

The risk of GVHD remains high following transplantation with currenttherapies. The methods and compositions disclosed herein may be used toinhibit graft versus host disease (GVHD) in a human patient. Theanti-CD137 ADCs may be used to selectively target activated T cells in apatient who will be receiving a transplant, such as a stem celltransplant. Anti-CD137 ADCs, as described herein, may also be used toreduce the risk of GVHD by targeting and depleting CD137 positive cellsin a human patient who is going to be or has already received atransplant, such as but not limited to, an HSC transplant. In certainembodiments, the compositions and methods disclosed herein are fortreating GVHD prior to appearance of symptoms of GVHD in a patientfollowing a transplantation therapy, e.g., allogeneic HSCs.

The methods described herein are also useful for preventing host versusgraft (HvG) reactions. An anti-CD137-ADC can also be used as animmunosuppressant to prevent host versus graft (HvG) reactions therebypreventing or reducing the risk of allogeneic graft failure. Use of ananti-CD137 ADC in a patient at risk for a HvG reaction would enableengraftment of donor cells with a greater degree of HLA-mismatch.Additional uses include tolerance induction in solid organ transplant,where host versus graft reactions are prevented or dampened by theCD137-ADC. These would include solid organ transplants done with orwithout hematopoietic stem cell transplants, including xeno-transplantswhere the organ is non-human in origin and/or genetically modified.

In one embodiment, an anti-CD137-ADC is used to prevent graft versusgraft (GvG) in the context of allogeneic transplants where two donorsare used. Examples include the use of 2 cord blood stem cell donors inadult and pediatric patients. Prevention of GvG would enable more rapidhematopoietic (e.g. neutrophil and platelet) reconstitutionpost-transplant as both stem cell sources would successfully engraft.

In some embodiments, the transplant is allogeneic. In some embodiments,the transplant is autologous.

In some embodiments, the transplant is a bone marrow transplant, aperipheral blood transplant, or a cord blood transplant.

In some embodiments, the transplant includes hematopoietic cells (e.g.,hematopoietic stem cells).

In any of the embodiments described herein, the transplant may be anysolid organ or skin transplant. In some embodiments, the transplant isselected from the group consisting of kidney transplant, hearttransplant, liver transplant, pancreas transplant, lung transplant,intestine transplant and skin transplant.

The methods described herein are useful for treating multiple sclerosis(MS). MS is a devastating autoimmune inflammatory disease of the centralnervous system. It is well accepted that the damage in the centralnervous system (CNS) results from an autoimmune attack against (auto)antigens within the myelin sheath. The mechanisms responsible for tissuedamage in MS involve the activation of self-reactive T cells, whichattack proteins in the myelin sheath. It is common for individuals toexperience the first signs between the ages of 15 and 50. Affectedindividuals encounter bouts of inflammatory demyelination producing theclassic course of the disease of exacerbation-remittance.

The methods described herein are also useful for treating human systemiclupus (SLE). SLE, or lupus, is a systemic chronic autoimmune diseasecharacterized by autoantibody production against self-antigens.Autoreactive B cells are driven by self-antigen, including antibodies todouble stranded DNA, to nuclear protein antigens and toribonucleoproteins. The factors that promote the loss of B celltolerance and drive autoantibody production are unknown. Systemic lupuscan affect almost any organ or system of the body. Systemic lupus mayinclude periods in which few, if any, symptoms are evident (“remission”)and other times when the disease becomes more active (“flare”).

The methods described herein are also useful for treating rheumatoidarthritis (RA). RA is a systemic autoimmune disease which initiallyattacks the synovium, a connective tissue membrane that lines the cavitybetween joints and secretes a lubricating fluid. Although the cause ofRA is unknown, infectious, genetic, and hormonal factors may contributeto the RA. RA is associated with abnormal immunity, as the joints ofpatients suffering from RA are severely infiltrated with leukocytes,such as macrophages and dendritic cells, and T and B cells. The diseasecan occur at any age, but the peak incidence of disease onset is betweenthe ages of 25 and 55. The incidence increases with age. The onset ofthe disease is usually gradual, with fatigue, morning stiffness lastingmore than one hour, diffuse muscular aches, loss of appetite, andweakness. Eventually, joint pain appears, with warmth, swelling,tenderness, and stiffness of the joint after inactivity.

The methods described herein are also useful for treating inflammatorybowel disease (IBD). Manifestations of IBD include ulcerative colitis,Crohn's disease, lymphocytic colitis, and collagenous colitis. IBD is aspontaneously relapsing, immunologically mediated disorder of thegastrointestinal tract, characterized by uncontrolled inflammation andpersistent activation of the mucosal immune system. CD4 T cells arebelieved to play a critical role in the pathogenesis of human IBD, dueto their influx into the inflamed mucosa.

The methods described herein are particularly useful for treatingpsoriasis. Psoriasis is a chronic inflammatory skin diseasecharacterized by red, scaly, raised plaques. Psoriasis is mediated by Tcells and associated elevation in cytokine levels leading to increasedcell division and aberrant differentiation. Psoriasis is a chronic,recurrent skin condition with varying degrees of severity and is alsoassociated with serious co-morbidities, including psoriatic arthritis,depression, malignancy, metabolic syndrome, cardiovascular morbidity andmortality and autoimmune diseases, such as inflammatory bowel disease(IBD).

The methods described herein are also useful for treating Type 1diabetes mellitus (Type 1 diabetes). Type 1 diabetes is a metabolicdisorder in humans that include juvenile onset patients that are notover-weight relative to their age and height, with rapid onset of thedisease at an early age, often before 30, although Type 1 diabetes canoccur at any age. Type 1 diabetes is considered to be a disease ofautoimmune etiology. CD4 and CD8 T cells have been implicated ascausative agents for damage to beta cells (insulin producing cells).

The methods described herein are also useful for treating otherautoimmune diseases including, but not limited to, acute disseminatedencephalomyelitis (ADEM), Addison's disease, alopecia universalis,ankylosing spondylitisis, antiphospholipid antibody syndrome (APS),aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune inner ear disease (AIED), autoimmune lymphoproliferativesyndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's disease,bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Crohn's disease, cicatrical pemphigoid, coeliacsprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome,Degos disease, discoid lupus, dysautonomia, endometriosis, essentialmixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture'ssyndrome, Grave's disease, Guillain-Barre syndrome (GBS), Hashimoto'sthyroiditis, Hidradenitis suppurativa, idiopathic and/or acutethrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy,interstitial cystitis, juvenile arthritis, Kawasaki's disease, lichenplanus, Lyme disease, Meniere disease, mixed connective tissue disease(MCTD), myasthenia gravis, neuromyotonia, opsoclonus myoclonus syndrome(OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, perniciousanemia, polychondritis, polymyositis and dermatomyositis, primarybiliary cirrhosis, polyarteritis nodosa, polyglandular syndromes,polymyalgia rheumatica, primary agammaglobulinemia, Raynaud phenomenon,Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren'ssyndrome, stiff person syndrome, Takayasu's arteritis, temporalarteritis (also known as “giant cell arteritis”), ulcerative colitis,uveitis, vasculitis, vitiligo, vulvodynia (“vulvar vestibulitis”), andWegener's granulomatosis.

The compositions and methods described herein can be used to treat avariety of disorders, including, without limitation, a non-malignanthemoglobinopathy (e.g., a hemoglobinopathy selected from the groupconsisting of sickle cell anemia, thalassemia, Fanconi anemia, andWiskott-Aldrich syndrome). Additionally or alternatively, thecompositions and methods described herein can be used to treat animmunodeficiency, such as a congenital immunodeficiency. Additionally oralternatively, the compositions and methods described herein can be usedto treat an acquired immunodeficiency (e.g., an acquiredimmunodeficiency selected from the group consisting of HIV and AIDS).The compositions and methods described herein can be used to treat ametabolic disorder (e.g., a metabolic disorder selected from the groupconsisting of glycogen storage diseases, mucopolysaccharidoses,Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromaticleukodystrophy). Additionally or alternatively, the compositions andmethods described herein can be used to treat a malignancy, such as ahematologic cancer (e.g., leukemia, lymphoma, multiple myeloma andmyelodysplastic syndrome), as well as other cancerous conditions,including neuroblastoma.

Additional disorders that can be treated by administration of thecompositions and methods described herein include adenosine deaminasedeficiency and severe combined immunodeficiency, hyper immunoglobulin Msyndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis,osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemiamajor, systemic sclerosis, systemic lupus erythematosus, multiplesclerosis, and juvenile rheumatoid arthritis.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsdescribed herein may be used, made, and evaluated, and are intended tobe purely exemplary of the invention and are not intended to limit thescope of what the inventors regard as their invention.

Example 1. Administration of an Anti-CD137 Antibody or ADC to a HumanPatient in Preparation for Hematopoietic Stem Cell Transplant Therapy

According to the methods disclosed herein, a physician of skill in theart can administer to the human patient an antibody, antigen-bindingfragment thereof, ADC, or soluble ligand capable of binding CD137, suchas an anti-CD137 antibody described herein. The antibody, fragmentthereof, ADC, or soluble ligand may be covalently conjugated to a toxin,such as a cytotoxic molecule described herein or known in the art, or anFc domain. For instance, an anti-CD137 antibody, antigen-bindingfragment thereof, ADC, or soluble ligand can be covalently conjugated toa cytotoxin, such as microtubule-binding agent, maytansine, amaytansinoid, an amatoxin, pseudomonas exotoxin A, deBouganin,diphtheria toxin, such as α-amanitin, saporin, an auristatin, ananthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, apyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof.

This conjugation can be performed using covalent bond-forming techniquesdescribed herein or known in the art. The antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, or drug-ligand conjugatecan subsequently be administered to the patient, for example, byintravenous administration, prior to transplantation of exogenoushematopoietic stem cells (such allogeneic hematopoietic stem cells) tothe patient.

The anti-CD137 antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered in an amountsufficient to reduce the quantity of host-reactive T cells, for example,by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more prior, atthe time of, or after the hematopoietic stem cell transplant therapy.The reduction in donor T cell count can be monitored using conventionaltechniques known in the art, such as by FACS analysis of cellsexpressing characteristic hematopoietic cell surface antigens in a bloodsample withdrawn from the patient. For instance, a physician of skill inthe art can withdraw a blood sample from the patient at various timepoints and determine the extent of donor CD137+ T cell reduction byconducting a FACS analysis to elucidate the relative concentrations of Tcells in the sample using antibodies that bind to donor T cell antigens.

The anti-CD137 antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered to the patientin an aqueous solution containing one or more pharmaceuticallyacceptable excipients, such as a viscosity-modifying agent. The aqueoussolution may be sterilized using techniques described herein or known inthe art. The antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered to the patientat a dosage of, for example, from 0.001 mg/kg to 100 mg/kg prior toadministration of a hematopoietic stem cell graft to the patient. Theantibody, antigen-binding fragment thereof, drug-antibody conjugate, ordrug-ligand conjugate can be administered to the patient at a time thatoptimally promotes engraftment of the exogenous hematopoietic stemcells, for instance, from 1 hour to 7 days (e.g., 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2days, 3 days, 4 days, 5 days, 6 days, or 7 days) or more prior to theadministration of the exogenous hematopoietic stem cell transplant. Forexample, the antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate may be administered about 3 daysprior to transplant. Alternatively, the antibody, antigen-bindingfragment thereof, drug-antibody conjugate, or drug-ligand conjugate canbe administered to the patient at a time that optimally promotesengraftment of the exogenous hematopoietic stem cells, for instance,concurrent with the administration of the exogenous hematopoietic stemcell transplant. Additionally, the antibody, antigen-binding fragmentthereof, drug-antibody conjugate, or drug-ligand conjugate can beadministered to the patient at a time that optimally promotesengraftment of the exogenous hematopoietic stem cells, for instance,from 1 hour to 10 days (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, or 10 days) or more after theadministration of the exogenous hematopoietic stem cell transplant. Forexample, the antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate may be administered about 3 to 4days after the transplant. The amount of antibody, antigen-bindingfragment thereof, drug-antibody conjugate, or drug-ligand conjugate canbe quantified, by methods known in the art, in the plasma of patients todetermine when the concentration of antibody, antigen-binding fragmentthereof, drug-antibody conjugate, or drug-ligand conjugate has reachedits maximum.

The patient may then receive an infusion (e.g., an intravenous infusion)of exogenous hematopoietic stem cells, such as from the same physicianthat administered the antibody or antigen-binding fragment thereof ordrug-antibody conjugate or from a different physician. The physician mayadminister the patient an infusion of autologous, syngeneic, orallogeneic hematopoietic stem cells, for instance, at a dosage of from1×10³ to 1×10⁹ CD34⁺ cells/kg. The physician may monitor the engraftmentof the hematopoietic stem cell transplant, for example, by withdrawing ablood sample from the patient and determining the increase inconcentration of hematopoietic stem cells or cells of the hematopoieticlineage (such as megakaryocytes, thrombocytes, platelets, erythrocytes,mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia,granulocytes, monocytes, osteoclasts, antigen-presenting cells,macrophages, dendritic cells, natural killer cells, T cells, and Bcells) following administration of the transplant. This analysis may beconducted, for example, from 1 hour to 6 months, or more, followinghematopoietic stem cell transplant therapy (e.g., 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or more). Afinding that the concentration of hematopoietic stem cells or cells ofthe hematopoietic lineage has increased (e.g., by 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,500%, or more) following the transplant therapy relative to theconcentration of the corresponding cell type prior to transplant therapyprovides one indication that treatment with the anti-CD137 antibody,antigen-binding fragment thereof, drug-antibody conjugate, ordrug-ligand conjugate has successfully promoted engraftment of thetransplanted hematopoietic stem cell graft.

Example 2. Generating Anti-CD137 Antibodies by Phage Display

An exemplary method for in vitro evolution of anti-CD137 antibodies foruse with the compositions and methods described herein is phage display.Phage display libraries can be created by making a designed series ofmutations or variations within a coding sequence for the CDRs of anantibody or the analogous regions of an antibody-like scaffold (e.g.,the BC, CD, and DE loops of ¹⁰Fn3 domains). The templateantibody-encoding sequence into which these mutations are introduced maybe, for example, a naive human germline sequence. These mutations can beperformed using standard mutagenesis techniques known in the art. Eachmutant sequence thus encodes an antibody corresponding to the templatesave for one or more amino acid variations. Retroviral and phage displayvectors can be engineered using standard vector construction techniquesknown in the art. P3 phage display vectors along with compatible proteinexpression vectors can be used to generate phage display vectors forantibody diversification.

The mutated DNA provides sequence diversity, and each transformant phagedisplays one variant of the initial template amino acid sequence encodedby the DNA, leading to a phage population (library) displaying a vastnumber of different but structurally related amino acid sequences. Dueto the well-defined structure of antibody hypervariable regions, theamino acid variations introduced in a phage display screen are expectedto alter the binding properties of the binding peptide or domain withoutsignificantly altering its overall molecular structure.

In a typical screen, a phage library may be contacted with and allowedto bind CD137 or an epitope thereof. To facilitate separation of bindersand non-binders, it is convenient to immobilize the target on a solidsupport. Phage bearing a CD137-binding moiety can form a complex withthe target on the solid support, whereas non-binding phage remain insolution and can be washed away with excess buffer. Bound phage can thenliberated from the target by changing the buffer to an extreme pH (pH 2or pH 10), changing the ionic strength of the buffer, addingdenaturants, or other known means.

The recovered phage can then be amplified through infection of bacterialcells, and the screening process can be repeated with the new pool thatis now depleted in non-binding antibodies and enriched for antibodiesthat bind CD137. The recovery of even a few binding phage is sufficientto amplify the phage for a subsequent iteration of screening. After afew rounds of selection, the gene sequences encoding the antibodies orantigen-binding fragments thereof derived from selected phage clones inthe binding pool are determined by conventional methods, thus revealingthe peptide sequence that imparts binding affinity of the phage to thetarget. During the panning process, the sequence diversity of thepopulation diminishes with each round of selection until desirablepeptide-binding antibodies remain. The sequences may converge on a smallnumber of related antibodies or antigen-binding fragments thereof. Anincrease in the number of phage recovered at each round of selection isan indication that convergence of the library has occurred in a screen.

Example 3. Producing Humanized Anti-CD137 Antibodies

Non-human antibodies that bind CD137 can be humanized, for instance,according to the following procedure. Consensus human antibody heavychain and light chain sequences are known in the art (see e.g., the“VBASE” human germline sequence database; Kabat et al. Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, 1991; Tomlinsonet al., J. Mol. Biol. 227:776-798, 1992; and Cox et al. Eur. J. Immunol.24:827-836, 1994, the disclosures of each of which are incorporatedherein by reference as they pertain to consensus human antibody heavychain and light chain sequences. Using established procedures, one ofskill in the art can identify the variable domain framework residues andCDRs of a consensus antibody sequence (e.g., by sequence alignment). Onecan substitute one or more CDRs of the heavy chain and/or light chainvariable domains of consensus human antibody with one or morecorresponding CDRs of a non-human antibody that binds CD137 in order toproduce a humanized antibody. This CDR exchange can be performed usinggene editing techniques described herein or known in the art.

One example of a variable domain of a consensus human antibody containsthe heavy chain variable domainEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS (SEQ ID NO:21) and the light chain variable domainDIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 22),identified in U.S. Pat. No. 6,054,297, the disclosure of which isincorporated herein by reference as it pertains to human antibodyconsensus sequences. The CDRs in the above sequences are shown in bold.

To produce humanized antibodies, one can recombinantly express apolynucleotide encoding the above consensus sequence in which one ormore variable region CDRs have been replaced with one or more variableregion CDR sequences of a non-human antibody that binds CD137. As theaffinity of the antibody for CD137 is determined primarily by the CDRsequences, the resulting humanized antibody is expected to exhibit anaffinity for CD137 that is about the same as that of the non-humanantibody from which the humanized antibody was derived. Methods ofdetermining the affinity of an antibody for a target antigen include,for instance, ELISA-based techniques described herein and known in theart, as well as surface plasmon resonance, fluorescence anisotropy, andisothermal titration calorimetry, among others.

Example 4. Administration of an Anti-CD137 Antibody or ADC to a HumanPatient at Risk for or Suffering from GVHD

According to the methods disclosed herein, a physician of skill in theart can administer to the human patient an antibody, antigen-bindingfragment thereof, ADC, or soluble ligand capable of binding CD137, suchas an anti-CD137 antibody or ADC described herein. The antibody,fragment thereof, ADC, or soluble ligand may be covalently conjugated toa toxin, such as a cytotoxic molecule described herein or known in theart, or an Fc domain. For instance, an anti-CD137 antibody,antigen-binding fragment thereof, or soluble ligand can be covalentlyconjugated to a cytotoxin, such as a microtubule-binding agent,maytansine, a maytansinoid, an amatoxin, pseudomonas exotoxin A,deBouganin, diphtheria toxin, such as α-amanitin, saporin, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof.

This conjugation can be performed using covalent bond-forming techniquesdescribed herein or known in the art. The antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, or drug-ligand conjugatecan subsequently be administered by intravenous administration, forexample, to a patient at risk for GVHD. The antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, or drug-ligand conjugatecan subsequently be administered by intravenous administration, forexample, to a patient suffering from GVHD. For example, the antibody,antigen-binding fragment thereof, or drug-antibody conjugate, ordrug-ligand conjugate can be administered to the patient prior, at thetime of, or after the transplantation of exogenous hematopoietic stemcells (such as allogeneic hematopoietic stem cells).

The anti-CD137 antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered in an amountsufficient to reduce the quantity of host-reactive T cells, for example,by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more followinghematopoietic stem cell transplant therapy. The reduction in donor Tcell count can be monitored using conventional techniques known in theart, such as by FACS analysis of cells expressing characteristichematopoietic cell surface antigens in a blood sample withdrawn from thepatient. For instance, a physician of skill in the art can withdraw ablood sample from the patient at various time points and determine theextent of donor CD137+ T cell reduction by conducting a FACS analysis toelucidate the relative concentrations of T cells in the sample usingantibodies that bind to donor T cell antigens.

The anti-CD137 antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered to the patientin an aqueous solution containing one or more pharmaceuticallyacceptable excipients, such as a viscosity-modifying agent. The aqueoussolution may be sterilized using techniques described herein or known inthe art. The antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered to the patientat a dosage of, for example, from 0.001 mg/kg to 100 mg/kg prior toadministration of a hematopoietic stem cell graft to the patient. Theantibody, antigen-binding fragment thereof, drug-antibody conjugate, ordrug-ligand conjugate can be administered to the patient at a time thatoptimally promotes prevention and treatment of GVHD, for instance, from1 hour to 7 days (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, or 7 days) or more prior to the administration of theexogenous hematopoietic stem cell transplant. For example, the antibody,antigen-binding fragment thereof, drug-antibody conjugate, ordrug-ligand conjugate may be administered about 3 days prior totransplant. Alternatively, the antibody, antigen-binding fragmentthereof, drug-antibody conjugate, or drug-ligand conjugate can beadministered to the patient at a time that optimally promotesengraftment of the exogenous hematopoietic stem cells, for instance,concurrent with the administration of the exogenous hematopoietic stemcell transplant. Additionally, the antibody, antigen-binding fragmentthereof, drug-antibody conjugate, or drug-ligand conjugate can beadministered to the patient at a time that optimally promotesengraftment of the exogenous hematopoietic stem cells, for instance,from 1 hour to 10 days (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, or 10 days) or more after theadministration of the exogenous hematopoietic stem cell transplant. Forexample, the antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate may be administered about 3 to 4days after the transplant. The amount of antibody, antigen-bindingfragment thereof, drug-antibody conjugate, or drug-ligand conjugate canbe quantified, by methods known in the art, in the plasma of patients todetermine when the concentration of antibody, antigen-binding fragmentthereof, drug-antibody conjugate, or drug-ligand conjugate has reachedits maximum.

The patient may then receive an infusion (e.g., an intravenous infusion)of exogenous hematopoietic stem cells, such as from the same physicianthat administered the antibody or antigen-binding fragment thereof ordrug-antibody conjugate, or from a different physician. The physicianmay administer the patient an infusion of autologous or allogeneichematopoietic stem cells, for instance, at a dosage of from 1×10³ to1×10⁹ CD34⁺ cells/kg.

A physician of skill in the art can evaluate the clinical manifestationsof GVHD after administering to the human patient an antibody,antigen-binding fragment thereof, ADC, or soluble ligand capable ofbinding CD137, such as an anti-CD137 antibody described herein.

Example 5. Administration of an Anti-CD137 Antibody or ADC to a HumanPatient that Develops an Autoimmune Disease as a Result of HematopoieticStem Cell Transplantation

According to the methods disclosed herein, a physician of skill in theart can administer to the human patient an antibody, antigen-bindingfragment thereof, ADC, or soluble ligand capable of binding CD137, suchas an anti-CD137 antibody or ADC described herein. The antibody,fragment thereof, or soluble ligand may be covalently conjugated to atoxin, such as a cytotoxic molecule described herein or known in theart, or an Fc domain. For instance, an anti-CD137 antibody,antigen-binding fragment thereof, or soluble ligand can be covalentlyconjugated to a cytotoxin, such as a microtubule-binding agent,maytansine, a maytansinoid, an amatoxin, pseudomonas exotoxin A,deBouganin, diphtheria toxin, such as α-amanitin, saporin, anauristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, aduocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, anindolinobenzodiazepine, and an indolinobenzodiazepine dimer, or avariant thereof.

This conjugation can be performed using covalent bond-forming techniquesdescribed herein or known in the art. The antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, or drug-ligand conjugatecan subsequently be administered by intravenous administration, forexample, to a patient at risk for an autoimmune disease (e.g., multiplesclerosis, rheumatoid arthritis, intestinal bowel disease, psoriasis,lupus, and Type 1 diabetes). For example, the antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, or drug-ligand conjugatecan be administered to the patient suffering from autoimmune diseasethat develops after the transplantation of exogenous hematopoietic stemcells (such as autologous or allogeneic hematopoietic stem cells).

The anti-CD137 antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered in an amountsufficient to reduce the quantity of host-reactive lymphocytes, forexample, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or moreprior to hematopoietic stem cell transplant therapy. The reduction indonor lymphocyte count can be monitored using conventional techniquesknown in the art, such as by FACS analysis of cells expressingcharacteristic hematopoietic cell surface antigens in a blood samplewithdrawn from the patient. For instance, a physician of skill in theart can withdraw a blood sample from the patient at various time pointsand determine the extent of CD137+ T cell reduction by conducting a FACSanalysis to elucidate the relative concentrations of T cells in thesample using antibodies that bind to T cell antigens. Efficacy againstautoimmune disease can be measured by assays known in the art (e.g.,autoantibody responses measurement from serum samples, and T cellproliferation in response to autoantigens).

The anti-CD137 antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered to the patientin an aqueous solution containing one or more pharmaceuticallyacceptable excipients, such as a viscosity-modifying agent. The aqueoussolution may be sterilized using techniques described herein or known inthe art. The antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered to the patientat a dosage of, for example, from 0.001 mg/kg to 100 mg/kg prior toadministration of a hematopoietic stem cell graft to the patient. Theantibody, antigen-binding fragment thereof, drug-antibody conjugate, ordrug-ligand conjugate can be administered to the patient at a time thatoptimally promotes prevention and treatment of autoimmune disease, forinstance, from 1 hour to 7 days (e.g., 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days,4 days, 5 days, 6 days, or 7 days) or more prior to the administrationof the exogenous hematopoietic stem cell transplant. For example, theantibody, antigen-binding fragment thereof, drug-antibody conjugate, ordrug-ligand conjugate may be administered about 3 days prior totransplant. Alternatively, the antibody, antigen-binding fragmentthereof, drug-antibody conjugate, or drug-ligand conjugate can beadministered to the patient at a time that optimally promotesengraftment of the exogenous hematopoietic stem cells, for instance,concurrent with the administration of the exogenous hematopoietic stemcell transplant. Additionally, the antibody, antigen-binding fragmentthereof, drug-antibody conjugate, or drug-ligand conjugate can beadministered to the patient at a time that optimally promotesengraftment of the exogenous hematopoietic stem cells, for instance,from 1 hour to 10 days (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, or 10 days) or more after theadministration of the exogenous hematopoietic stem cell transplant. Forexample, the antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate may be administered about 3 to 4days after the transplant. The amount of antibody, antigen-bindingfragment thereof, drug-antibody conjugate, or drug-ligand conjugate canbe quantified, by methods known in the art, in the plasma of patients todetermine when the concentration of antibody, antigen-binding fragmentthereof, drug-antibody conjugate, or drug-ligand conjugate has reachedits maximum.

The patient may then receive an infusion (e.g., an intravenous infusion)of exogenous hematopoietic stem cells, such as from the same physicianthat administered the antibody or antigen-binding fragment thereof ordrug-antibody conjugate or from a different physician. The physician mayadminister the patient an infusion of autologous or allogeneichematopoietic stem cells, for instance, at a dosage of from 1×10³ to1×10⁹ CD34+ cells/kg.

A physician of skill in the art can evaluate the clinical manifestationsof autoimmune disease after administering to the human patient anantibody, antigen-binding fragment thereof, ADC, or soluble ligandcapable of binding CD137, such as an anti-CD137 antibody or ADCdescribed herein.

Example 6. Administration of an Anti-CD137 Antibody to a Human Patientat Risk or Suffering from an Autoimmune Disease

According to the methods disclosed herein, a physician of skill in theart can administer to the human patient an antibody, antigen-bindingfragment thereof, ADC, or soluble ligand capable of binding CD137, suchas an anti-CD137 antibody or ADC described herein. The antibody,fragment thereof, or soluble ligand may be covalently conjugated to atoxin, such as a cytotoxic molecule described herein or known in theart, or an Fc domain.

This conjugation can be performed using covalent bond-forming techniquesdescribed herein or known in the art. The antibody, antigen-bindingfragment thereof, or drug-antibody conjugate, or drug-ligand conjugatecan subsequently be administered by intravenous administration, forexample, to a patient at risk for autoimmune disease (e.g., multiplesclerosis, rheumatoid arthritis, intestinal bowel disease, psoriasis,lupus, and Type 1 diabetes). The antibody, antigen-binding fragmentthereof, or drug-antibody conjugate, or drug-ligand conjugate cansubsequently be administered by intravenous administration, for example,to a patient suffering from autoimmune disease (e.g., multiplesclerosis, rheumatoid arthritis, intestinal bowel disease, psoriasis,lupus, and Type 1 diabetes).

The anti-CD137 antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered in an amountsufficient to reduce the quantity of host-reactive lymphocytes, forexample, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.The reduction in donor lymphocyte count can be monitored usingconventional techniques known in the art, such as by FACS analysis ofcells expressing characteristic hematopoietic cell surface antigens in ablood sample withdrawn from the patient. For instance, a physician ofskill in the art can withdraw a blood sample from the patient at varioustime points and determine the extent of CD137+ T cell reduction byconducting a FACS analysis to elucidate the relative concentrations of Tcells in the sample using antibodies that bind to T cell antigens.Efficacy against autoimmune disease can be measured by assays known inthe art (e.g., autoantibody responses measurement from serum samples,and T cell proliferation in response to autoantigens).

The anti-CD137 antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered to the patientin an aqueous solution containing one or more pharmaceuticallyacceptable excipients, such as a viscosity-modifying agent. The aqueoussolution may be sterilized using techniques described herein or known inthe art. The antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be administered to the patientat a dosage of, for example, from 0.001 mg/kg to 100 mg/kg prior toadministration of a hematopoietic stem cell graft to the patient. Theantibody, antigen-binding fragment thereof, drug-antibody conjugate, ordrug-ligand conjugate can be administered to the patient at a time thatoptimally promotes prevention and treatment of autoimmune disease. Theamount of antibody, antigen-binding fragment thereof, drug-antibodyconjugate, or drug-ligand conjugate can be quantified, by methods knownin the art, in the plasma of patients to determine when theconcentration of antibody, antigen-binding fragment thereof,drug-antibody conjugate, or drug-ligand conjugate has reached itsmaximum.

A physician of skill in the art can evaluate the clinical manifestationsof autoimmune disease after administering to the human patient anantibody, antigen-binding fragment thereof, or soluble ligand capable ofbinding CD137, such as an anti-CD137 antibody or ADC described herein.

Example 7. In Vitro Cell Line Binding Assay

Jurkat cells (i.e., an immortalized human T lymphocyte cell line)characterized by stable, over-expression of hCD137 were plated at 20,000cells/well and stained with a titration of primary murine anti-CD137antibody BBK2 (BBK2-mIgG1) for 4 hours at 4° C. Secondary anti-mouseAF488 stain, at a constant amount, was added for 30 minutes at 4° C.After washing, plates were run on a flow cytometer and binding ofBBK2-mIgG1 (and the negative control, i.e., mIgG1) was determined basedon geometric mean fluorescence intensity in the AF488 channel. Resultsfrom these assays are provided in FIGS. 1A and B.

As shown in FIGS. 1A and 1B, the murine BBK2 antibody binds to human Tcells (i.e. CD137-expressing Jurkat cells), with an EC₅₀=35 pM.

Example 8. In Vitro Analysis of an Anti-CD137-Amanitin Antibody DrugConjugate (ADC) Using an In Vitro T Cell Killing Assay

Cryopreserved negatively-selected primary human T cells were thawed andstimulated with anti-CD3/anti-CD28 beads (Invitrogen) at a bead:cellratio of 0.5:1. At the start of the assay, 2e4 T cells were seeded perwell of a 384 well plate and ADCs were added to the wells at variousconcentrations between 0.003 nm and 30 nm before being placed in anincubator with 37° C. and 5% CO₂. Following 4 days of culture, cellswere analyzed by flow cytometry. Cells were stained with a viabilitymarker Live/Dead Yellow (Invitrogen) and run on a volumetric flowcytometer. Numbers of viable, activated cells (FIG. 2A) and viable,non-activated cells (FIG. 2B) were determined by FSC vs SSC. Anon-specific human IgG conjugated to amanitin (hIgG-amanitin) served asa negative control. Thus control was compared to two different ADCs: 1)chimeric anti-CD137 antibody BBK2 conjugated to amanitin(CD137-Amanitin); and 2) an ADC including an antibody specific a T-cellantigen conjugated to Amanitin (anti-Tcell-Amanitin). Theanti-Tcell-Amanitin ADC served as a positive control as it was expectedto bind and kill both activated and non-activated T cells.

As shown in FIGS. 2A and 2B, the anti-CD137-amanitin ADC (i.e.,“CD137-Amanitin) specifically killed activated T cells and did notappreciably kill non-activated (resting, steady-state) T cells. Inaddition, the positive control (i.e., the “anti-Tcell-Amanitin” ADC),which was an ADC that specifically targeted both activated andnon-activated T cells, killed both activated and non-activated T cells.

Example 9. Analysis of the Prevention of GVHD Using a Xeno-GVHD MouseModel

The ability of an anti-CD137-amanitin ADC to prevent the formation oroccurrence of GVHD was assessed in vivo using a xeno-GVHD mouse model.Female, 6-8-week-old NSG mice were irradiated (200 cGy) and transplantedthe following day with 6×10⁶ human peripheral blood mononuclear cells(PBMCs) to generate the GVHD mouse model. One day later, animals weredosed (at 3 mg/kg) with an anti-CD137-amanitin ADC (i.e.,“CD137-Amanitin”) or with various control reagents (i.e., buffer alone(“PBS”), an anti-CD137 antibody (“CD137 Naked”), or an amanitin-basedADC that is not specific to CD137 (“Isotype-Amanitin”)). The anti-CD137antibody used in this example in the ADCs and as a naked antibody waschimeric BBK2.

Animals were followed closely daily after dosing for signs of GVHDand/or decreased body conditions including, but not limited to, hunchedposture, ruffled fur, weight loss, and/or limited activity. Animals withsevere body condition concerns, or those animals that showed weightloss >20%, were sacrificed and their tissues were analyzed for humancells. Peripheral blood and spleens of mice were stained with a cocktailof antibodies, including hCD45, mCD45, hCD3, and hCD137 antibodies, redblood cell were lysed, and analyzed by flow cytometry. The number ofhuman T cells in the blood were defined as hCD45+CD3+ and normalized toinput blood volume. The percent survival as a function of dayspost-treatment is provided in FIG. 3. The number of human T cells in theperipheral blood as a function of days post-transplant is provided inFIG. 4.

As demonstrated in FIG. 3, the animals treated with a single dose of theanti-CD137-amanitin ADC (“CD137-Amanitin”) showed essentially completeprevention of GVHD, even at 80 days post-transplant, while the animalstreated with a control (i.e., PBS, an anti-CD137 antibody (naked), andisotype-amanitin) all died within 11 to 13 days post-transplant. Theseresults also indicate that the anti-CD137-amanitin ADC waswell-tolerated in all animals and that a single dose of theanti-CD137-amanitin ADC was sufficient to completely prevent GVHD (asopposed to requiring multiple doses). As demonstrated in FIG. 4, nohuman T cells were detectable in the peripheral blood of mice over aperiod of at least 70 days after the transplant in anti-CD137-amanitintreated mice, while animals treated with a control were characterized ashaving human T cells detectable in the peripheral blood of mice severaldays post-transplant, indicating the development of GVHD in the animalstreated with a control.

Example 10. Determination of Engraftment Rates and Steady-State T CellDepletion in a hNSG-SGM3 Mouse Model

Female, humanized NSG-SGM3 mice were evaluated for baseline humanhematopoietic (overall and T-cell) engraftment rates in the peripheralblood by flow cytometry using hCD45, mCD45, hCD3, and hCD137 antibodies.Mice were randomized and then treated with CD137-Amanitin ADC (chimericBBK2-Amanitin ADC), Isotype-Amanitin ADC, or an anti-T cell AmanitinADC, each at a dose of 3 mg/kg. Engraftment and T cell depletion in theperiphery of the mice was measured at day 5, day 9, day 14, day 22 andday 27 post treatment (FIGS. 5A and 5B). Peripheral blood was stained toevaluate changes in chimerism and T cell numbers. Equivalent amounts ofblood were run on a volumetric flow cytometer and absolute counts weredetermined based on event numbers. Decreases in engraftment and T cellnumbers was normalized to baseline values.

The results in FIGS. 5A and 5B indicate that the anti-CD137-amanitin ADCwas well-tolerated in this mouse model. Further, engraftment and T cellfrequency (after normalizing to baseline) remained near baseline levels,with a moderate to transient decrease in engraftment and T cellfrequency for the anti-CD137-amanitin ADC treated mice. These dataindicate that there is generally a lack of T cell depletion insteady-state humanized NSG mice, which indicates that the immunefunction can be preserved in anti-CD137-amanitin ADC treated micebecause most of the T cells are not depleted. To the contrary, micetreated with an ADC that specifically targets an alternate T cell marker(“anti-Tcell-Amanitin”) demonstrated complete ablation of engraftmentand T cell numbers.

The heavy and light chain amino acid sequences of chimeric BBK2described in the above examples are set forth in SEQ ID NOs: 23 and 24,respectively.

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from theinvention that come within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

1. A method of depleting a population of CD137 positive cells in a humanpatient having graft-versus-host disease (GVHD) or at risk fordeveloping GVHD, the method comprising administering to the patient aneffective amount of an antibody capable of binding CD137, wherein theantibody is conjugated to a cytotoxin via a linker, and wherein thecytotoxin is an amatoxin.
 2. The method of claim 1, wherein the amatoxinis represented by formula (IV)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody, wherein Am comprises exactly one R_(C) substituent.
 3. Themethod of claim 1, wherein the amatoxin is an amanitin.
 4. The method ofclaim 3, wherein the amanitin is selected from the group consisting ofα-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide,amanullin, amanullinic acid, and proamanullin.
 5. The method of claim 1,wherein the method comprises administering the antibody to the patientprior to the patient receiving a transplant comprising hematopoieticstem cells.
 6. The method of claim 1, wherein the method comprisesadministering the antibody to the patient concomitantly with the patientreceiving a transplant comprising hematopoietic stem cells.
 7. Themethod of claim 1, wherein the method comprises administering theantibody to the patient after the patient receives a transplantcomprising hematopoietic stem cells.
 8. The method of claim 7, themethod comprising administering the antibody to the patient about 1 hourto 10 days after the patient receives a transplant comprisinghematopoietic stem cells.
 9. The method of claim 1, wherein the antibodycomprises a heavy chain variable region comprising a CDR1, a CDR2, and aCDR3 having an amino acid sequence as set forth in SEQ ID NOs: 25, 26,and 27, respectively, and comprising a light chain variable regioncomprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence asset forth in SEQ ID NOs: 29, 30, and 31, respectively.
 10. The method ofclaim 5, wherein the transplant is selected from the group consisting ofan allogeneic transplant, a bone marrow transplant, a peripheral bloodtransplant, and a cord blood transplant.
 11. A method of treatinggraft-versus-host disease (GVHD) in a human patient in need thereof, themethod comprising administering an anti-CD137 antibody drug conjugate(ADC) to the human patient such that GVHD is treated, wherein the ADCcomprises an anti-CD137 antibody linked to a cytotoxin, wherein thecytotoxin is an amatoxin.
 12. The method of claim 11, wherein the methodcomprises administering the ADC to the patient prior to the patientreceiving a transplant comprising hematopoietic stem cells.
 13. Themethod of claim 11, the method comprising administering the ADC to thepatient about three days prior to the patient receiving a transplantcomprising hematopoietic stem cells.
 14. The method of claim 11, whereinthe method comprises administering the ADC to the patient concomitantwith the patient receiving a transplant comprising hematopoietic stemcells.
 15. The method of claim 11, wherein the method comprisesadministering the ADC to the patient after the patient receives atransplant comprising hematopoietic stem cells.
 16. The method of claim11, wherein the anti-CD137 antibody comprises a heavy chain variableregion comprising a CDR1, a CDR2, and a CDR3 having an amino acidsequence as set forth in SEQ ID NOs: 25, 26, and 27, respectively, andcomprising a light chain variable region comprising a CDR1, a CDR2, anda CDR3 having an amino acid sequence as set forth in SEQ ID NOs: 29, 30,and 31, respectively.
 17. The method of claim 11, wherein the amatoxinis represented by formula (IV)

wherein R₁ is H, OH, OR_(A), or OR_(C); R₂ is H, OH, OR_(B), or OR_(C);R_(A) and R_(B), together with the oxygen atoms to which they are bound,combine to form an optionally substituted 5-membered heterocyclolalkylgroup; R₃ is H, R_(C), or R_(D); R₄, R₅, R₆, and R₇ are eachindependently H, OH, OR_(C), OR_(D), R_(C), or R_(D); R₈ is OH, NH₂,OR_(C), OR_(D), NHR_(C), or NR_(C)R_(D); R₉ is H, OH, OR_(C), or OR_(D);X is —S—, —S(O)—, or —SO₂—; R_(C) is -L-Z; R_(D) is optionallysubstituted C₁-C₆ alkyl, optionally substituted C₁-C₆ heteroalkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆heteroalkenyl, optionally substituted C₂-C₆ alkynyl, optionallysubstituted C₂-C₆ heteroalkynyl, optionally substituted cycloalkyl,optionally substituted heterocycloalkyl, optionally substituted aryl, oroptionally substituted heteroaryl; L is optionally substituted C₁-C₆alkylene, optionally substituted C₁-C₆ heteroalkylene, optionallysubstituted C₂-C₆ alkenylene, optionally substituted C₂-C₆heteroalkenylene, optionally substituted C₂-C₆ alkynylene, optionallysubstituted C₂-C₆ heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted arylene, or optionally substituted heteroarylene; and Z is achemical moiety formed from a coupling reaction between a reactivesubstituent present on L and a reactive substituent present within theantibody, wherein Am comprises exactly one R_(C) substituent.
 18. Themethod of claim 11, wherein the amatoxin is an amanitin.
 19. The methodof claim 18, wherein the amanitin is selected from the group consistingof α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide,amanullin, amanullinic acid, and proamanullin.
 20. A method of depletingallo-reactive T cells in a human patient who received an allogenictransplant, the method comprising administering an anti-CD137 ADC to thehuman patient such that allo-reactive T cells are depleted, wherein theADC comprises an anti-CD137 antibody linked to a cytotoxin.
 21. Themethod of claim 20, wherein the transplant is selected from the groupconsisting of a bone marrow transplant, a peripheral blood transplantand a cord blood transplant.
 22. The method of claim 20, wherein thetransplant comprises hematopoietic cells.
 23. The method of claim 20,wherein the hematopoietic stem cells or progeny thereof maintainhematopoietic stem cell functional potential after two or more daysfollowing transplantation of the hematopoietic stem cells into thepatient.
 24. The method of claim 20, wherein the cytotoxin is an RNApolymerase inhibitor.
 25. The method of claim 24, wherein the RNApolymerase inhibitor is an amatoxin.
 26. The method of claim 20, whereinthe anti-CD137 antibody comprises a heavy chain variable regioncomprising a CDR1, a CDR2, and a CDR3 having an amino acid sequence asset forth in SEQ ID NOs: 25, 26, and 27, respectively, and comprising alight chain variable region comprising a CDR1, a CDR2, and a CDR3 havingan amino acid sequence as set forth in SEQ ID NOs: 29, 30, and 31,respectively.
 27. An antibody drug conjugate (ADC) comprising ananti-CD137 antibody conjugated to a cytotoxin via a linker, wherein theantibody comprises a heavy chain variable region comprising a CDR1, aCDR2, and a CDR3 having an amino acid sequence as set forth in SEQ IDNOs: 25, 26, and 27, respectively, and comprising a light chain variableregion comprising a CDR1, a CDR2, and a CDR3 having an amino acidsequence as set forth in SEQ ID NOs: 29, 30, and 31, respectively. 28.The ADC of claim 27, wherein the heavy chain variable region comprisesan amino acid sequence as set forth in SEQ ID NO: 28 and the light chainvariable region comprises an amino acid sequence as set forth in SEQ IDNO:
 32. 29. A pharmaceutical composition comprising the ADC of claim 27,and a pharmaceutically acceptable carrier.